Cytokine protein family

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for zcyto20, zcyto21, zcyto22, zycto24, and zcyto25 proteins which are most closely related to interferon-α at the amino acid sequence level. The receptor for this protein family is a class II cytokine receptor. The present invention includes methods of reducing viral infections and increasing monocyte counts. The present invention also includes antibodies to the zcyto20 polypeptides, and methods of producing the polynucleotides and polypeptides.

REFERENCE TO RELATED APPLICATIONS

The present application is divisional of U.S. patent application Ser.No. 11/266,626, filed Nov. 3, 2005, which is a divisional of U.S. patentapplication Ser. No. 10/127,816, now U.S. Pat. No. 7,038,032, filed Apr.19, 2002, which is a continuation-in-part of U.S. patent applicationSer. No. 09/895,834, filed Jun. 29, 2001, now U.S. Pat. No. 6,927,040,and claims the benefit of U.S. Patent Application Ser. No. 60/285,424,filed Apr. 20, 2001, U.S. Patent Application Ser. No. 60/285,408, filedApr. 20, 2001, U.S. Patent Application Ser. No. 60/286,482, filed Apr.25, 2001, U.S. Patent Application Ser. No. 60/341,050, filed Oct. 22,2001, and U.S. Patent Application Ser. No. 60/341,105, filed Oct. 22,2001, all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Cellular differentiation of multicellular organisms is controlled byhormones and polypeptide growth factors. These diffusible moleculesallow cells to communicate with each other and act in concert to formtissues and organs, and to repair and regenerate damaged tissue.Examples of hormones and growth factors include the steroid hormones,parathyroid hormone, follicle stimulating hormone, the interferons, theinterleukins, platelet derived growth factor, epidermal growth factor,and granulocyte-macrophage colony stimulating factor, among others.

Hormones and growth factors influence cellular metabolism by binding toreceptor proteins. Certain receptors are integral membrane proteins thatbind with the hormone or growth factor outside the cell, and that arelinked to signaling pathways within the cell, such as second messengersystems. Other classes of receptors are soluble intracellular molecules.

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

Cytokines play important roles in the regulation of hematopoiesis andimmune responses, and can influence lymphocyte development. The humanclass II cytokine family includes interferon-α (IFN-α) subtypes,interferon-β (IFN-β), interferon-γ (IFN-γ), IL-10, IL-19 (U.S. Pat. No.5,985,614), MDA-7 (Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-20(Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-22 (Xie et al., J.Biol. Chem. 275, 31335-31339, (2000)), and AK-155 (Knappe et al., J.Virol. 74, 3881-3887, (2000)). Most cytokines bind and transduce signalsthrough either Class I or Class II cytokine receptors. Members of humanclass II cytokine receptor family include interferon-αR1 (IFN-αR1),interferon-γ-R2 (IFN-γ-R2), interferon-γ R1 (IFN-γ R1), interferon-γR2(IFN-γR2), IL-10R (Liu et al., J. Immunol. 152, 1821-1829, (1994)),CRF2-4 (Lutfalla et al. Genomics 16, 366-373, (1993)), IL-20Rβ (Blumberget al., Cell 104, 9-19, (2001)) (also known as zcytor7 (U.S. Pat. No.5,945,511) and CRF2-8 (Kotenko et al., Oncogene 19, 2557-2565, (2000)),IL-20Rβ (Blumberg et al., ibid, (2001)) (also known as DIRS1 (PCT WO99/46379)), IL-22RA1 (IL-22 receptor-α1, submitted to HUGO for approval)(also known as IL-22R (Xie et al., J. Biol. Chem. 275, 31335-31339,(2000)), zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-9 (Kotenko et al.,Oncogene 19, 2557-2565, (2000)), and tissue factor.

Class II cytokine receptors are typically heterodimers composed of twodistinct receptor chains, the α and β receptor subunits (Stahl et al.,Cell 74, 587-590, (1993)). In general, the α subunits are the primarycytokine binding proteins, and the β subunits are required for formationof high affinity binding sites, as well as for signal transduction. Anexception is the IL-20 receptor in which both subunits are required forIL-20 binding (Blumberg et al., ibid, (2001)).

The class II cytokine receptors are identified by a conservedcytokine-binding domain of about 200 amino acids (D200) in theextracellular portion of the receptor. This cytokine-binding domain iscomprised of two fibronectin type III (FnIII) domains of approximately100 amino acids each (Bazan J. F. Proc. Natl. Acad. Sci. USA 87,6934-6938, (1990); Thoreau et al., FEBS Lett. 282, 16-31, (1991)). EachFnIII domain contains conserved Cys, Pro, and Trp residues thatdetermine a characteristic folding pattern of seven β-strands similar tothe constant domain of immunoglobulins (Uze et al., J. InterferonCytokine Res. 15, 3-26, (1995)). The conserved structural elements ofthe class II cytokine receptor family make it possible to identify newmembers of this family on the basis of primary amino acid sequencehomology. Previously we have successfully identified two new members ofclass II cytokine receptor family, zcytor7 (U.S. Pat. No. 5,945,511)(also known as IL-20R α (Blumberg et al., ibid, (2001)) and zcytor11(U.S. Pat. No. 5,965,704) (also known as IL-22R (Blumberg et al., ibid,(2001)), using this approach. Identification of additional novel membersof the class II cytokine receptor family is of interest becausecytokines play a vital role in regulating biological responses.

IL-22, also known as IL-TIF (IL-10-related T cell-derived induciblefactor) (Dumoutier et al., J. Immunology 164, 1814-1819, (2000)), is arecently described IL-10 homologue. Mouse IL-22 was originallyidentified as a gene induced by IL-9 in T cells and mast cells in vitro(Dumoutier et al., J. Immunology 164, 1814-1819, (2000)). Acute phasereactant induction activity was observed in mouse liver upon IL-22injection, and IL-22 expression was rapidly induced afterlipopolysaccharide (LPS) injection, suggesting that IL-22 contributes tothe inflammatory response in vivo (Dumoutier et al., Proc. Natl. Acad.Sci. U.S.A. 97, 10144-10149, (2000)).

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. The interleukins mediate a variety ofinflammatory pathologies. Central to an immune response is the T cell,which produce many cytokines and adaptive immunity to antigens.Cytokines produced by the T cell have been classified as type 1 and type2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokinesinclude IL-2, IFN-γ, LT-α, and are involved in inflammatory responses,viral immunity, intracellular parasite immunity and allograft rejection.Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and areinvolved in humoral responses, helminth immunity and allergic response.Shared cytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α.There is some evidence to suggest that Type 1 and Type 2 producing Tcell populations preferentially migrate into different types of inflamedtissue.

Of particular interest, from a therapeutic standpoint, are theinterferons (reviews on interferons are provided by De Maeyer and DeMaeyer-Guignard, “Interferons,” in The Cytokine Handbook, 3^(rd)Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), andby Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages158-188 (John Wiley & Sons 1998)). Interferons exhibit a variety ofbiological activities, and are useful for the treatment of certainautoimmune diseases, particular cancers, and the enhancement of theimmune response against infectious agents, including viruses, bacteria,fungi, and protozoa. To date, six forms of interferon have beenidentified, which have been classified into two major groups. Theso-called “type I” interferons include interferon-α, interferon-β,interferon-ω, interferon-δ, and interferon-τ. Currently, interferon-γand one subclass of interferon-α are the only type II interferons.

Type I interferons, which are thought to be derived from the sameancestral gene, have retained sufficient similar structure to act by thesame cell surface receptor. The α-chain of the human interferon-α/βreceptor comprises an extracellular N-terminal domain, which has thecharacteristics of a class II cytokine receptor. Interferon-γ does notshare significant homology with the type I interferons or with the typeII interferon-α subtype, but shares a number of biological activitieswith the type I interferons.

In humans, at least 16 non-allelic genes code for different subtypes ofinterferon-α, while interferons β and ω are encoded by single genes.Type I interferon genes are clustered in the short arm of chromosome 9.Unlike typical structural human genes, interferon-α, interferon-β, andinterferon-ω lack introns. A single gene for human interferon-γ islocalized on chromosome 12 and contains three introns. To date,interferon-τ has been described only in cattle and sheep, whileinterferon-δ has been described only in pigs.

Clinicians are taking advantage of the multiple activities ofinterferons by using the proteins to treat a wide range of conditions.For example, one form of interferon-α has been approved for use in morethan 50 countries for the treatment of medical conditions such as hairycell leukemia, renal cell carcinoma, basal cell carcinoma, malignantmelanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis,mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitisC, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.Food and Drug Administration has approved the use of interferon-β totreat multiple sclerosis, a chronic disease of the nervous system.Interferon-γ is used to treat chronic granulomatous diseases, in whichthe interferon enhances the patient's immune response to destroyinfectious bacterial, fungal, and protozoal pathogens. Clinical studiesalso indicate that interferon-γ may be useful in the treatment of AIDS,leishmaniasis, and lepromatous leprosy.

The demonstrated in vivo activities of the cytokine family illustratethe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. The present inventionaddresses these needs by providing a new cytokine that stimulates cellsof the hematopoietic cell lineage, as well as related compositions andmethods.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention includes a genus of polynucleotide and polypeptidemolecules that have functional and structural similarity to theinterferons. In this new family, which includes molecules designatedzcyto20(SEQ ID NOS: 1 and 2), zcyto21 (SEQ ID NOS:4 and 5), zcyto22 (SEQID NOS:6 and 7), zcyto24 (SEQ ID NOS:8 and 9), zcyto25 (SEQ ID NOS: 10and 11), zcyto20, 21, and 22 are human sequences and zcyto24 and 25 aremouse sequences. Homology within the family at the nucleotide and aminoacid levels is shown in Table 1, ranging from approximately 72% to 98%at the nucleotide level, and 51% to 97% at the amino acid level.

TABLE 1 nucleotide sequence identity zcyto20 zcyto22 zcyto21 zcyto24zcyto25 rat protein zcyto20 100 98.2 72.9 74.0 72.1 73.4 se- zcyto2296.0 100 73.0 73.9 71.9 72.9 quence zcyto21 66.5 67.5 100 64.9 62.9 64.6identity zcyto24 62.7 63.7 51.7 100 97.2 90.3 zcyto25 59.8 60.8 48.893.6 100 88.4

Table 2 is an illustration of the sequence identity between zcyto20,zcyto21, zcyto22, IFNα, IFNβ, IFNγ, and IL10 at the amino acid level.

TABLE 2 amino acid sequence identity Zcyto20 Zcyto22 Zcyto21 IFNα IFNβIFNγ IL10 Zcyto20 100 Zcyto21 81 100 Zcyto22 96 74 100 IFNα 17 16 17 100IFNβ 14 13 14 31 100 IFNγ 4 4 4 7 5 100 IL10 13 12 14 7 5 8 100

All the members of the family have been shown to bind to the same classII cytokine receptor, designated zcytor19 receptor. Furthermore, certainbiological activities have been shown to be exhibited by each moleculein the family. These activities include, for example, antiviralactivities and increasing circulating myeloid cell levels. While notwanting to be bound by theory, these molecules appear to all signalthrough zcytor19 receptor via the same pathway.

Zcyto20 gene encodes a polypeptide of 205 amino acids, as shown in SEQID NO:2. The signal sequence for Zcyto20 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 21 (Ala) of SEQ IDNO:2. The mature peptide for Zcyto20 begins at amino acid residue 22(Val).

Zcyto21 gene encodes a polypeptide of 200 amino acids, as shown in SEQID NO:5. The signal sequence for Zcyto21 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 19 (Ala) of SEQ IDNO:5. The mature peptide for Zcyto21 begins at amino acid residue 20(Gly). Zcyto21 has been described in PCT application WO 02/02627.

Zcyto22 gene encodes a polypeptide of 205 amino acids, as shown in SEQID NO:7. The signal sequence for Zcyto22 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 21 (Ala) of SEQ IDNO:7. The mature peptide for Zcyto22 begins at amino acid residue 22(Val).

Zcyto24 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:9. Zcyto24 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:9. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

Zcyto25 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:11. Zcyto25 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:11. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

The Zcyto20, Zcyto21 and Zcyto22 genes have been mapped to humanchromosome 19q13.13. Based on the discovery of these genes, this regionof chromosome 19 has been identified as comprising a cluster ofinterferon-like genes. Further indication that this is new family ofgenes is identification of a syntenic cluster of genes on the mousechromosome 7, zcyto24 (SEQ ID NO: 8) and zcyto25 (SEQ ID NO: 10).

As described below, the present invention provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acidresidues 22 to 205 of SEQ ID NO:2 or amino acid residues 1 to 205 of SEQID NO:2, or some fragment thereof. The present invention also includes apolypeptide that further comprises a signal secretory sequence thatresides in an amino-terminal position relative to the first amino acidsequence, wherein the signal secretory sequence comprises amino acidresidues 1 to 21 of the amino acid sequence of SEQ ID NO:2.

In another embodiment, the present invention provides isolatedpolypeptides having an amino acid sequence that is at least 70%, atleast 80%, or at least 90%, 95%, 96%, 97%, 98% or 99% identical toeither amino acid residues 22 to 205 of SEQ ID NO:7 or amino acidresidues 1 to 205 of SEQ ID NO:7. The present invention also includes apolypeptide that further comprises a signal secretory sequence thatresides in an amino-terminal position relative to the first amino acidsequence, wherein the signal secretory sequence comprises amino acidresidues 1 to 21 of the amino acid sequence of SEQ ID NO:7.

In general, cytokines, like erythropoietin (EPO), are predicted to havea four-alpha helix structure, with helices A, C and D being mostimportant in ligand-receptor interactions, and are more highly conservedamong members of the family. However, the interferons (INF), andinterferon-alpha and interferon-tau in particular, are characterized assix helix bundles. EPO helix A is equivalent to helix A of zcyto20; EPOhelix B is equivalent to helix C of zcyto20; EPO helix C is equivalentto helix D of zcyto20, and EPO helix D is equivalent to helix F ofzcyto20. Thus, the loop between the AB loop, and CD loop of EPO isexpanded in zcyto20 to contain short helices B and E of zcyto20. Thehelical structures of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 aresimilar to the six-helix structure found in the interferons. Boundariesof secondary structures in proteins are generally defined in accordancewith a range of PHI and PSI angles of the protein chain backbone from a3-dimensional model of the protein. Models may be constructed from, forexample, x-ray crystallography or NMR data, or homology modeling basedon a solved structure. Depending on techniques used, includingconditions for crystal formation and flexible NMR solution structuredetermination, boundaries of these secondary structures may be slightlyaltered. Thus, those skilled in the art will recognize that the helicalboundaries, and secondary structures in general, depending on theenvironment, may shift by as much as 2, 3, 4, or more residues, buthelical regions are essentially as described below. (See, Brandon andToosze, Introduction to Protein Structure, Garland Publishing Co, inc.New York, 1991; Anderson, et. al., Structure, 10(2):175-84. 2002.)

Zcyto20 helices are predicted as follow: helix A is defined by aminoacid residues 52 (Ala) to 66 (Leu); helix B by amino acid residues 78(Arg) to 87 (Val); helix C by amino acid residues 91 (Pro) to 108 (Thr);helix D by amino acid residues 116 (Val) to 138 (Ser); helix E by aminoacid residues 151 (Thr) to 172 (Lys); and helix F by amino acid residues177 (Gly) to 197 (Cys); as shown in SEQ ID NO: 2. Four cysteine residuesare conserved between Zcyto20, Zcyto21, and INF-α. In addition, Zcyto20has 3 additional cysteines. The cysteine at amino acid residue 204, mayform an intermolecular disulfide bond, in particular to form homodimerswith additional Zcyto20 molecules. Further analysis of Zcyto20 based onmultiple alignments predicts that cysteines at amino acid residues 37and 136; 69 and 197; and 71 and 178 (as shown in SEQ ID NO: 2) will formintramolecular disulfide bonds. The corresponding polynucleotidesencoding the Zcyto20 polypeptide regions, domains, motifs, residues andsequences described herein are as shown in SEQ ID NO:1.

Zcyto21 helices are predicted as follows: helix A is defined by aminoacid residues 49 (Ser) to 63 (Leu); helix B by amino acid residues 76(Asn) to 84 (Val); helix C by amino acid residues 89 (Val) to 104 (Ala);helix D by amino acid residues 111 (Glu) to 133 (Gln); helix E by aminoacid residues 137 (Thr) to 158 (Lys); and helix F by amino acid residues163 (Gly) to 189 (Leu); as shown in SEQ ID NO: 5. The cysteine residuesare conserved between Zcyto21, Zcyto21, and INF-α, and may form anintermolecular disulfide bond, in particular to form homodimers withadditional Zcyto21 molecules. Further analysis of Zcyto21 based onmultiple alignments predicts that cysteines at amino acid residues 34and 131, and 68 and 164, will form intramolecular disulfide bonds. Thecysteine at residue 190 is free, and may form an intermoleculardisulfide association. The corresponding polynucleotides encoding theZcyto21 polypeptide regions, domains, motifs, residues and sequencesdescribed herein are as shown in SEQ ID NO:4.

Zcyto22 helices are predicted as follow: helix A is defined by aminoacid residues 52 (Ala) to 66 (Leu); helix B by amino acid residues 78(Arg) to 87 (Val); helix C by amino acid residues 91 (Pro) to 108 (Thr);helix D by amino acid residues 116 (Val) to 138 (Ser); helix E by aminoacid residues 151 (Thr) to 172 (Lys); and helix F by amino acid residues177 (Gly) to 197 (Cys); as shown in SEQ ID NO: 7. Four cysteine residuesare conserved between Zcyto22, Zcyto21, and INF-α. In addition, Zcyto22has 3 additional cysteines. The cysteine at amino acid residue 204, mayform an intermolecular disulfide bond, in particular to form homodimerswith additional Zcyto22 molecules. Further analysis of Zcyto22 based onmultiple alignments predicts that cysteines at amino acid residues 37and 136; 69 and 197; and 71 and 178 (as shown in SEQ ID NO: 7) will formintramolecular disulfide bonds. The corresponding polynucleotidesencoding the Zcyto22 polypeptide regions, domains, motifs, residues andsequences described herein are as shown in SEQ ID NO:6.

Conserved cysteines for zcyto24 are shown at residues 44, 78, 141, and175 of SEQ ID NO: 9. Further analysis of zcyto24 based on multiplealignments predicts that disulfide bonds will be formed betweencysteines at amino acid residues 44 and 141; 78 and 175; (as shown inSEQ ID NO: 9). The corresponding polynucleotides encoding the zcyto24polypeptide regions, domains, motifs, residues and sequences describedherein are as shown in SEQ ID NO:9. The predicted helices in zcyto24 (asshown in SEQ ID NO:9) are: residues 59-73 (helix A); residues 85-94(helix B); residues 98-115 (helix C); residues 121-143 (helix D);residues 147-169 (helix E); residues 174-194 (helix F).

Conserved cysteines for zcyto25 are shown at residues 44, 78, 141, and175 of SEQ ID NO: 11. Further analysis of zcyto25 based on multiplealignments predicts that disulfide bonds will be formed betweencysteines at amino acid residues 44 and 141; 78 and 175; (as shown inSEQ ID NO: 11). The corresponding polynucleotides encoding the zcyto25polypeptide regions, domains, motifs, residues and sequences describedherein are as shown in SEQ ID NO:11. The predicted helices in zcyto25(as shown in SEQ ID NO:11) are: residues 59-73 (helix A); residues 85-94(helix B); residues 98-115 (helix C); residues 121-143 (helix D);residues 147-169 (helix E); residues 174-194 (helix F).

Detailed mutational analysis of murine IL-2 (Zurawski et al., EMBO J.12:5113-5119, 1993) shows residues in helices A and C are important forbinding to IL-2Rβ; critical residues are Asp₃₄, Asn₉₉, and Asn₁₀₃.Multiple residues within murine IL-2 loop A/B and helix B are importantfor IL-2Rα binding, while only a single residue, Gln₁₄₁ in helix D, isvital for binding with IL-2Rα. Similarly, helices A and C are sites ofinteraction between IL-4 and IL-4Rα (the structurally similar toIL-2Rα), and residues within helix D are vital for IL-2Rα interaction(Wang et al., Proc. Natl. Acad. Sci. USA 94:1657-1662, 1997; Kruse etal., EMBO J. 11:3237-3244, 1992). In particular, the mutation Tyr₁₂₄ toAsp in human IL-4 creates an antagonist, which binds with IL-4Rα but notIL-2Rα and therefore cannot signal (Kruse et al. ibid. 1992).

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices, and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Studies using CNTFand IL-6 demonstrated that a CNTF helix can be exchanged for theequivalent helix in IL-6, conferring CTNF-binding properties to thechimera. Thus, it appears that functional domains of four-helicalcytokines are determined on the basis of structural homology,irrespective of sequence identity, and can maintain functional integrityin a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999).Therefore, the helical domains of zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 will be useful for preparing chimeric fusion molecules,particularly with other interferons to determine and modulate receptorbinding specificity. Of particular interest are fusion proteins thatcombine helical and loop domains from interferons and cytokines such asINF-α, IL-10, human growth hormone.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 have been shown to form acomplex with the orphan receptor designated zcytor19. Zcytor19 isdescribed in a commonly assigned patent application PCT/US01/44808.Zcyto22, zcyto21, and zcyto24 have been shown to bind or signal throughzcytor19 as well, further supporting that zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 are members of the same family of cytokines.Zcytor19 receptor is a class II cytokine receptor. Class II cytokinereceptors usually bind to four-helix-bundle cytokines. For example,interleukin-10 and the interferons bind receptors in this class (e.g.,interferon-gamma receptor, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains).

Class II cytokine receptors are characterized by the presence of one ormore cytokine receptor modules (CRM) in their extracellular domains.Other class II cytokine receptors include zcytor11 (commonly owned U.S.Pat. No. 5,965,704), CRF2-4 (Genbank Accession No. Z17227), IL-10R(Genbank Accession Nos. U00672 and NM_(—)001558), DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511), and tissue factor. Zcytor19,like all known class II receptors except interferon-alpha/beta receptoralpha chain, has only a single class II CRM in its extracellular domain.

Analysis of a human cDNA clone encoding Zcytor19 (SEQ ID NO:26) revealedan open reading frame encoding 520 amino acids (SEQ ID NO:27) comprisinga secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ IDNO:27) and a mature zcytor19 cytokine receptor polypeptide (residues 21(Arg) to 520 (Arg) of SEQ ID NO:27) an extracellular ligand-bindingdomain of approximately 206 amino acid residues (residues 21 (Arg) to226 (Asn) of SEQ ID NO:27), a transmembrane domain of approximately 23amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO:27),and an intracellular domain of approximately 271 amino acid residues(residues 250 (Lys) to 520 (Arg) of SEQ ID NO:27). Within theextracellular ligand-binding domain, there are two fibronectin type IIIdomains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:27, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:27, and thesecond fibronectin type III domain comprises residues 125 (Pro) to 223(Pro) of SEQ ID NO:27. Thus, a polypeptide comprising amino acids 21(Arg) to 223 (Pro) of SEQ ID NO:27 is considered a ligand bindingfragment. In addition as typically conserved in class II receptors,there are conserved Tryptophan residues comprising residues 43 (Trp) and68 (Trp) as shown in SEQ ID NO:27, and conserved Cysteine residues atpositions 74, 82, 195, 217 of SEQ ID NO:27.

In addition, a human cDNA clone encoding a Zcytor19 variant with a 30amino acid deletion was identified. This zcytor19 variant (as shown inSEQ ID NO:23) comprises an open reading frame encoding 491 amino acids(SEQ ID NO:24) comprising a secretory signal sequence (residues 1 (Met)to 20 (Gly) of SEQ ID NO:24) and a mature zcytor19 cytokine receptorpolypeptide (residues 21 (Arg) to 491 (Arg) of SEQ ID NO:24) anextracellular ligand-binding domain of approximately 206 amino acidresidues (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:24, atransmembrane domain of approximately 23 amino acid residues (residues227 (Trp) to 249 (Trp) of SEQ ID NO:24), and an intracellular domain ofapproximately 242 amino acid residues (residues 250 (Lys) to 491 (Arg)of SEQ ID NO:24). Within the extracellular ligand-binding domain, thereare two fibronectin type III domains and a linker region. The firstfibronectin type III domain comprises residues 21 (Arg) to 119 (Tyr) ofSEQ ID NO:24, the linker comprises residues 120 (Leu) to 124 (Glu) ofSEQ ID NO:24, and the second fibronectin type III domain is short, andcomprises residues 125 (Pro) to 223 (Pro) of SEQ ID NO:24. Thus, apolypeptide comprising amino acids 21 (Arg) to 223 (Pro) of SEQ ID NO:24is considered a ligand binding fragment. In addition as typicallyconserved in class II receptors, there are conserved Tryptophan residuescomprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:24, andconserved Cysteine residues at positions 74, 82, 195, 217 of SEQ IDNO:24.

A truncated soluble form of the zcytor19 receptor mRNA appears to benaturally expressed. Analysis of a human cDNA clone encoding thetruncated soluble Zcytor19 (SEQ ID NO:28) revealed an open reading frameencoding 211 amino acids (SEQ ID NO:29) comprising a secretory signalsequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:29) and a maturetruncated soluble zcytor19 receptor polypeptide (residues 21 (Arg) to211 (Ser) of SEQ ID NO:29) a truncated extracellular ligand-bindingdomain of approximately 143 amino acid residues (residues 21 (Arg) to163 (Trp) of SEQ ID NO:29), no transmembrane domain, but an additionaldomain of approximately 48 amino acid residues (residues 164 (Lys) to211 (Ser) of SEQ ID NO:29). Within the truncated extracellularligand-binding domain, there are two fibronectin type III domains and alinker region. The first fibronectin type III domain comprises residues21 (Arg) to 119 (Tyr) of SEQ ID NO:29, the linker comprises residues 120(Leu) to 124 (Glu) of SEQ ID NO:29, and the second fibronectin type IIIdomain comprises residues 125 (Pro) to 163 (Trp) of SEQ ID NO:29. Thus,a polypeptide comprising amino acids 21 (Arg) to 163 (Trp) of SEQ IDNO:29 is considered a ligand binding fragment. In addition as typicallyconserved in class II receptors, there are conserved Tryptophan residuescomprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:29, andconserved Cysteine residues in this truncated soluble form of thezcytor19 receptor are at positions 74, and 82 of SEQ ID NO:29.

Zcytor19 receptor is a member of the same receptor subfamily as theclass II cytokine receptors, and receptors in this subfamily mayassociate to form homodimers that transduce a signal. Several members ofthe subfamily (e.g., receptors that bind interferon, IL-10, IL-19, andIL-TIF) combine with a second subunit (termed a β-subunit) to bindligand and transduce a signal. However, in many cases, specificβ-subunits associate with a plurality of specific cytokine receptorsubunits. For example, class II cytokine receptors, such as, zcytor11(U.S. Pat. No. 5,965,704) and CRF2-4 receptor heterodimerize to bind thecytokine IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al., J.Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med.187:571-578, 1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4cDNA); Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000). IL-10βreceptor is believed to be synonymous with CRF2-4 (Dumoutier, L. et al.,Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; Liu Y et al, J. Immunol.152; 1821-1829, 1994 (IL-10R cDNA). Therefore, one could expect thatzcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 would bind eithermonomeric, homodimeric, heterodimeric and multimeric zcytor19 receptors.Experimental evidence has identified CRF2-4 (SEQ ID NOS: 40 and 41) asthe putative binding partner for zcytor19 which provides further supportthat zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 play an importantrole in the immunomodulatory system, affecting physiologies such as theinnate immune system and the inflammatory response system.

Localizing the expression of a receptor for a ligand/receptor pair mayhave significance for identifying the target cell or tissue at which theligand acts. This is particularly useful when the receptor/ligandcomplex involves a heterodimeric receptor in which one of the subunitsis expressed widely and another the of the subunits is expressed in alimited manner, either spatially or temporally restricted. Using in situhybridization expression of zcytor19 has been identified in a skincarcinoma sample, where the cancerous granular epithelium was stronglypositive, while no positive signal is observed in normal skin. Othertissues identified as expressing zcytor19 included fetal liver, wheresignal was observed in a mixed population of mononuclear cells insinusoid spaces; in lung expression was observed in type II alveolarepithelium; and in macrophage-like mononuclear cells in the interstitialtissue. Northern analysis of zcytor19 identified expression of a ˜4.5 kbtranscript which was in greatest in heart, skeletal muscle, pancreas,and prostate tissue, in addition to in the Burkitt's lymphoma (RAJI)cell line and SW-480 colorectal carcinoma cell line.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode the zcyto20, zcyto21, zcyto22, zcyto24,and zcyto25 polypeptides disclosed herein. Those skilled in the art willreadily recognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. SEQ ID NO:3 is a degenerate DNA sequence that encompasses allDNAs that encode the zcyto20 polypeptide of SEQ ID NO:2. Those skilledin the art will recognize that the degenerate sequence of SEQ ID NO:3also provides all RNA sequences encoding SEQ ID NO:2 by substituting Ufor T. Thus, zcyto20 polypeptide-encoding polynucleotides comprisingnucleotide 1 or 64 to nucleotide 615 of SEQ ID NO:3 and their RNAequivalents are contemplated by the present invention. Table 3 setsforth the one-letter codes used within SEQ ID NO:3 to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, with A being complementary to T, and Gbeing complementary to C.

SEQ ID NO:46 is a degenerate DNA sequence that encompasses all DNAs thatencode the zcyto22 polypeptide of SEQ ID NO:7. Those skilled in the artwill recognize that the degenerate sequence of SEQ ID NO:46 alsoprovides all RNA sequences encoding SEQ ID NO:7 by substituting U for T.Thus, zcyto22 polypeptide-encoding polynucleotides comprising nucleotide1 or 64 to nucleotide 615 of SEQ ID NO:46 and their RNA equivalents arecontemplated by the present invention. Table 3 sets forth the one-lettercodes used within SEQ ID NO:46 to denote degenerate nucleotidepositions. “Resolutions” are the nucleotides denoted by a code letter.“Complement” indicates the code for the complementary nucleotide(s). Forexample, the code Y denotes either C or T, and its complement R denotesA or G, with A being complementary to T, and G being complementary to C.

TABLE 3 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 4.

TABLE 4 One Amino Letter Degenerate Acid Code Condons Codon Cys C TGCTGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN ProP CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGTGGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAGCAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAGAAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTGYTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp WTGG TGG Ter • TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2 and SEQ ID NO:7. Variant sequences can bereadily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 3). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNOS:3 and 46 serves as a template for optimizing expression ofpolynucleotides in various cell types and species commonly used in theart and disclosed herein. Sequences containing preferential codons canbe tested and optimized for expression in various species, and testedfor functionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 RNA. Such tissues and cells are identified by Northern blotting(Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), or by screeningconditioned medium from various cell types for activity on target cellsor tissue. Once the activity or RNA producing cell or tissue isidentified, total RNA can be prepared using guanidinium isothiocyanateextraction followed by isolation by centrifugation in a CsCl gradient(Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA isprepared from total RNA using the method of Aviv and Leder (Proc. Natl.Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) is preparedfrom poly(A)⁺ RNA using known methods. In the alternative, genomic DNAcan be isolated. Polynucleotides encoding zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 polypeptides are then identified and isolated by,for example, hybridization or PCR.

A full-length clones encoding zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 can be obtained by conventional cloning procedures.Complementary DNA (cDNA) clones are preferred, although for someapplications (e.g., expression in transgenic animals) it may bepreferable to use a genomic clone, or to modify a cDNA clone to includeat least one genomic intron. Methods for preparing cDNA and genomicclones are well known and within the level of ordinary skill in the art,and include the use of the sequence disclosed herein, or parts thereof,for probing or priming a library. Expression libraries can be probedwith antibodies to Zcytor19 receptor fragments, or other specificbinding partners.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptidesfrom other mammalian species, including murine, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. Orthologs ofhuman zcyto20, zcyto21, and zcyto22 can be cloned using information andcompositions provided by the present invention in combination withconventional cloning techniques. For example, a cDNA can be cloned usingmRNA obtained from a tissue or cell type that expresses zcyto20,zcyto21, and zcyto22 as disclosed herein. Suitable sources of mRNA canbe identified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. Zcyto20-, zcyto21-, and zcyto22-encodingcDNA can then be isolated by a variety of methods, such as by probingwith a complete or partial human cDNA or with one or more sets ofdegenerate probes based on the disclosed sequences. A cDNA can also becloned using the polymerase chain reaction, or PCR (Mullis, U.S. Pat.No. 4,683,202), using primers designed from the representative humanzcyto20 sequence disclosed herein. Within an additional method, the cDNAlibrary can be used to transform or transfect host cells, and expressionof the cDNA of interest can be detected with an antibody to zcyto20polypeptide, binding studies or activity assays. Similar techniques canalso be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NOS:1, 4, and 6, respectively, represent single alleles of humanzcyto20, zcyto21, and zcyto22 bands, and that allelic variation andalternative splicing are expected to occur. Allelic variants of thissequence can be cloned by probing cDNA or genomic libraries fromdifferent individuals according to standard procedures. Allelic variantsof the DNA sequence shown in SEQ ID NO:1, 4 and 6, including thosecontaining silent mutations and those in which mutations result in aminoacid sequence changes, are within the scope of the present invention, asare proteins which are allelic variants of SEQ ID NO:2, 5, and 7. cDNAsgenerated from alternatively spliced mRNAs, which retain the propertiesof the zcyto20, zcyto21, and zcyto22 polypeptides, are included withinthe scope of the present invention, as are polypeptides encoded by suchcDNAs and mRNAs. Allelic variants and splice variants of these sequencescan be cloned by probing cDNA or genomic libraries from differentindividuals or tissues according to standard procedures known in theart.

The present invention also provides reagents which will find use indiagnostic applications. For example, the zcyto20, zcyto21, and zcyto22genes, probes comprising zcyto20, zcyto21, and zcyto22 DNA or RNA or asubsequence thereof can be used to determine if the zcyto20, zcyto21,and zcyto22 gene is present on a human chromosome, such as chromosome19, or if a gene mutation has occurred. Zcyto20, zcyto21, and zcyto22are located at the q13.13 region of chromosome 19. Detectablechromosomal aberrations at the zcyto20, zcyto21, and zcyto22 gene locusinclude, but are not limited to, aneuploidy, gene copy number changes,loss of heterogeneity (LOH), translocations, insertions, deletions,restriction site changes and rearrangements. Such aberrations can bedetected using polynucleotides of the present invention by employingmolecular genetic techniques, such as restriction fragment lengthpolymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.;Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

For example, Delague et al., (Am. J. Hum. Genet. 67: 236-243, 2000)identified that Charcot-Marie-Tooth disease is localized to 19q13.1-13.3(Delague et al., Am. J. Hum. Genet. 67: 236-243, 2000).

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or assistance in genetic counseling.As such, the inventive anti-zcyto20 antibodies, polynucleotides, andpolypeptides can be used for the detection of zcyto20 polypeptide, mRNAor anti-zcyto20 antibodies, thus serving as markers and be directly usedfor detecting or genetic diseases or cancers, as described herein, usingmethods known in the art and described herein. Further, zcyto20,zcyto21, and zcyto22 polynucleotide probes can be used to detectabnormalities or genotypes associated with chromosome 19q13.13 deletionsand translocations associated with human diseases or othertranslocations involved with malignant progression of tumors or other19q13.13 mutations, which are expected to be involved in chromosomerearrangements in malignancy; or in other cancers. Similarly, zcyto20polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 19q13.13 trisomy and chromosome lossassociated with human diseases or spontaneous abortion. Thus, zcyto20,zcyto21, and zcyto22 polynucleotide probes can be used to detectabnormalities or genotypes associated with these defects.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Analytical probes will be generally at least 20 nt in length,although somewhat shorter probes can be used (e.g., 14-17 nt). PCRprimers are at least 5 nt in length, preferably 15 or more, morepreferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, azcyto20 polynucleotide probe may comprise an entire exon or more. Exonsare readily determined by one of skill in the art by comparing zcyto20,zcyto21, and zcyto22 sequences (SEQ ID NOS:1, 4, and 6, respectively)with the genomic DNA for zcyto20, zcyto21, and zcyto22. In general, thediagnostic methods used in genetic linkage analysis, to detect a geneticabnormality or aberration in a patient, are known in the art. Mostdiagnostic methods comprise the steps of (a) obtaining a genetic samplefrom a potentially diseased patient, diseased patient or potentialnon-diseased carrier of a recessive disease allele; (b) producing afirst reaction product by incubating the genetic sample with a zcyto20polynucleotide probe wherein the polynucleotide will hybridize tocomplementary polynucleotide sequence, such as in RFLP analysis or byincubating the genetic sample with sense and antisense primers in a PCRreaction under appropriate PCR reaction conditions; (iii) Visualizingthe first reaction product by gel electrophoresis and/or other knownmethod such as visualizing the first reaction product with a zcyto20polynucleotide probe wherein the polynucleotide will hybridize to thecomplementary polynucleotide sequence of the first reaction; and (iv)comparing the visualized first reaction product to a second controlreaction product of a genetic sample from wild type patient. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the diseased orpotentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the zcyto20 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1, thecomplement of SEQ ID NO:1, or an RNA equivalent thereof. Such methods ofshowing genetic linkage analysis to human disease phenotypes are wellknown in the art. For reference to PCR based methods in diagnostics seesee, generally, Mathew (ed.), Protocols in Human Molecular Genetics(Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methodsand Applications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Mutations associated with the zcyto20, zcyto21, and zcyto22 locus can bedetected using nucleic acid molecules of the present invention byemploying standard methods for direct mutation analysis, such asrestriction fragment length polymorphism analysis, short tandem repeatanalysis employing PCR techniques, amplification-refractory mutationsystem analysis, single-strand conformation polymorphism detection,RNase cleavage methods, denaturing gradient gel electrophoresis,fluorescence-assisted mismatch analysis, and other genetic analysistechniques known in the art (see, for example, Mathew (ed.), Protocolsin Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (HumanPress, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases(Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols forMutation Detection (Oxford University Press 1996), Birren et al. (eds.),Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor LaboratoryPress 1998), Dracopoli et al. (eds.), Current Protocols in HumanGenetics (John Wiley & Sons 1998), and Richards and Ward, “MolecularDiagnostic Testing,” in Principles of Molecular Medicine, pages 83-88(Humana Press, Inc. 1998)). Direct analysis of an zcyto20 gene for amutation can be performed using a subject's genomic DNA. Methods foramplifying genomic DNA, obtained for example from peripheral bloodlymphocytes, are well-known to those of skill in the art (see, forexample, Dracopoli et al. (eds.), Current Protocols in Human Genetics,at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).

Within embodiments of the invention, isolated zcyto20-encoding nucleicacid molecules can hybridize under stringent conditions to nucleic acidmolecules having the nucleotide sequence of SEQ ID NO:1, to nucleic acidmolecules having the nucleotide sequence of nucleotides 64 to 618 of SEQID NO:1, or to nucleic acid molecules having a nucleotide sequencecomplementary to SEQ ID NO:1. In general, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Withinembodiments of the invention, isolated zcyto22-encoding nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules having the nucleotide sequence of SEQ ID NO:6, to nucleic acidmolecules having the nucleotide sequence of nucleotides 64 to 618 of SEQID NO:6, or to nucleic acid molecules having a nucleotide sequencecomplementary to SEQ ID NO:6.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

It is well within the abilities of one skilled in the art to adapt theseconditions for use with a particular polynucleotide hybrid. The T_(m)for a specific target sequence is the temperature (under definedconditions) at which 50% of the target sequence will hybridize to aperfectly matched probe sequence. Those conditions which influence theT_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al, Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×−2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant zcyto20, zcyto21, and zcyto22 polypeptides hybridizewith a nucleic acid molecule having the nucleotide sequence of SEQ IDNOS:1, 4, and 6, respectively (or its complement) under stringentwashing conditions, in which the wash stringency is equivalent to0.5×−2×SSC with 0.1% SDS at 55-65° C., including 0.5×SSC with 0.1% SDSat 55° C., or 2×SSC with 0.1% SDS at 65° C. One of skill in the art canreadily devise equivalent conditions, for example, by substituting SSPEfor SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×−0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantzcyto20 polypeptide hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×−0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 polypeptides that have a substantially similarsequence identity to the polypeptides of SEQ ID NOS:2, 5, 7, 9, 11,respectively, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70%, at least 80%, at least 90%, at least 95%, or greater than 95%sequence identity to the sequences shown in SEQ ID NOS:2, 5, 7, 9, 11,respectively, or their orthologs. The present invention also includespolypeptides that comprise an amino acid sequence having at least 70%,at least 80%, at least 90%, at least 95% or greater than 95%, 96%, 97%,98%, 99% sequence identity to the sequence of amino acid residues 1 to205 or 21 to 205 of SEQ ID NO:2 or SEQ ID NO:7. The present inventionfurther includes nucleic acid molecules that encode such polypeptides.Methods for determining percent identity are described below.

The present invention also contemplates variant zcyto20, zcyto21, andzcyto22 nucleic acid molecules that can be identified using twocriteria: a determination of the similarity between the encodedpolypeptide with the amino acid sequence of SEQ ID NOS:2, 5, 7, 9, 11,respectively, and/or a hybridization assay, as described above. Suchzcyto20 variants include nucleic acid molecules: (1) that hybridize witha nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:1,4, 6, 8, 10, respectively (or its complement) under stringent washingconditions, in which the wash stringency is equivalent to 0.5×−2×SSCwith 0.1% SDS at 55-65° C.; or (2) that encode a polypeptide having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the amino acid sequence of SEQ ID NO:2.Alternatively, zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 variantscan be characterized as nucleic acid molecules: (1) that hybridize witha nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:1,4, 6, 8, 10, respectively (or its complement) under highly stringentwashing conditions, in which the wash stringency is equivalent to0.1×−0.2×SSC with 0.1% SDS at 50-65° C.; and (2) that encode apolypeptide having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the amino acid sequence ofSEQ ID NOS:2, 5, 7, 9, 11, respectively.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 4 (amino acids are indicated by the standard one-lettercodes).

$\frac{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}}{\begin{bmatrix}{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}\mspace{14mu} {the}} \\{{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}} \\{{sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$

TABLE 5 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zcyto20. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant zcyto20, zcyto21, and zcyto22 polypeptides or polypeptides withsubstantially similar sequence identity are characterized as having oneor more amino acid substitutions, deletions or additions. These changesare preferably of a minor nature, that is conservative amino acidsubstitutions (see Table 5) and other substitutions that do notsignificantly affect the folding or activity of the polypeptide; smalldeletions, typically of one to about 30 amino acids; and amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or anaffinity tag. The present invention thus includes polypeptides of fromabout 154 to 235 amino acid residues that comprise a sequence that is atleast 70%, preferably at least 90%, and more preferably 95%, 96%, 97%,98%, 99% or more identical to the corresponding region of SEQ ID NO:2.Polypeptides comprising affinity tags can further comprise a proteolyticcleavage site between the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polypeptide and the affinity tag. Preferred such sites include thrombincleavage sites and factor Xa cleavage sites.

TABLE 6 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 polypeptides so as to minimize disruption of higherorder structure essential to biological activity. For example, where thezcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptide comprises oneor more helices, changes in amino acid residues will be made so as notto disrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichroism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the zcyto20 protein sequence asshown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad.Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 andTriquier et al., Protein Engineering 11: 153-169, 1998). The profile isbased on a sliding six-residue window. Buried G, S, and T residues andexposed H, Y, and W residues were ignored. For example, in zcyto20,hydrophilic regions include amino acid residues 169 (Glu) to 174 (Glu)of SEQ ID NO: 2, amino acid residues 54 (Lys) to 59 (Ala) of SEQ ID NO:2, amino acid residues 53 (Phe) to 58 (Asp) of SEQ ID NO: 2, amino acidresidues 168 (Gln) to 173 (Lys) of SEQ ID NO: 2, and amino acid residues154 (Pro) to 159 (Arg) of SEQ ID NO: 2.

A Hopp/Woods hydrophilicity profile of the zcyto22 protein sequence asshown in SEQ ID NO:7 can be generated (Hopp et al., Proc. Natl. Acad.Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 andTriquier et al., Protein Engineering 11: 153-169, 1998). The profile isbased on a sliding six-residue window. Buried G, S, and T residues andexposed H, Y, and W residues were ignored. For example, in zcyto22,hydrophilic regions include amino acid residues 169 (Glu) to 174 (Glu)of SEQ ID NO: 7, amino acid residues 54 (Lys) to 59 (Ala) of SEQ ID NO:7, amino acid residues 53 (Phe) to 58 (Asp) of SEQ ID NO: 7, amino acidresidues 168 (Gln) to 173 (Lys) of SEQ ID NO: 7, and amino acid residues154 (Pro) to 159 (Arg) of SEQ ID NO: 7.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 polypeptide, so as not to disrupt the overall structural andbiological profile. Of particular interest for replacement arehydrophobic residues selected from the group consisting of Val, Leu andIle or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between INF-α and members of the familyof zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 (as shown in Tables 1and 2). Using methods such as “FASTA” analysis described previously,regions of high similarity are identified within a family of proteinsand used to analyze amino acid sequence for conserved regions. Analternative approach to identifying a variant polynucleotide on thebasis of structure is to determine whether a nucleic acid moleculeencoding a potential variant zcyto20, zcyto21, zcyto22, zcyto24, andzcyto25 gene can hybridize to a nucleic acid molecule having thenucleotide sequence of SEQ ID NOS:1, 4, 6, 8, or 10 as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalor biochemical activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides and nucleic acidmolecules encoding such functional fragments. A “functional” zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 or fragment thereof as definedherein is characterized by its proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 antibody or zcytor19 receptor(either soluble or immobilized). As previously described herein,zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides arecharacterized by a six-helical-bundle. Thus, the present inventionfurther provides fusion proteins encompassing: (a) polypeptide moleculescomprising one or more of the helices described above; and (b)functional fragments comprising one or more of these helices. The otherpolypeptide portion of the fusion protein may be contributed by anotherhelical-bundle cytokine or interferon, such as INF-α, or by a non-nativeand/or an unrelated secretory signal peptide that facilitates secretionof the fusion protein.

The zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides of thepresent invention, including full-length polypeptides, biologicallyactive fragments, and fusion polypeptides can be produced according toconventional techniques using cells into which have been introduced anexpression vector encoding the polypeptide. As used herein, “cells intowhich have been introduced an expression vector” include both cells thathave been directly manipulated by the introduction of exogenous DNAmolecules and progeny thereof that contain the introduced DNA. Suitablehost cells are those cell types that can be transformed or transfectedwith exogenous DNA and grown in culture, and include bacteria, fungalcells, and cultured higher eukaryotic cells. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 polypeptide is operably linked to other genetic elementsrequired for its expression, generally including a transcriptionpromoter and terminator, within an expression vector. The vector willalso commonly contain one or more selectable markers and one or moreorigins of replication, although those skilled in the art will recognizethat within certain systems selectable markers may be provided onseparate vectors, and replication of the exogenous DNA may be providedby integration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

To direct a zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptideinto the secretory pathway of a host cell, a secretory signal sequence(also known as a leader sequence, prepro sequence or pre sequence) isprovided in the expression vector. The secretory signal sequence may bethat of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25, or may bederived from another secreted protein (e.g., t-PA; see, U.S. Pat. No.5,641,655) or synthesized de novo. The secretory signal sequence isoperably linked to the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25DNA sequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Cultured mammalian cells can be used as hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K¹, ATCC No. CCL 61; or CHODG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter. Expression vectors for use in mammaliancells include pZP-1 and pZP-9, which have been deposited with theAmerican Type Culture Collection, Manassas, Va. USA under accessionnumbers 98669 and 98668, respectively, and derivatives thereof.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected non-293 cells under conditions wherethe cells are not rapidly dividing, the cells can produce proteins forextended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. In an alternativemethod, adenovirus vector-infected 293 cells can be grown as adherentcells or in suspension culture at relatively high cell density toproduce significant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins can also be effectivelyobtained.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV)according to methods known in the art. Within a preferred method,recombinant baculovirus is produced through the use of atransposon-based system described by Luckow et al. (J. Virol.67:4566-4579, 1993). This system, which utilizes transfer vectors, iscommercially available in kit form (Bac-to-Bac™ kit; Life Technologies,Rockville, Md.). The transfer vector (e.g., pFastBac1™; LifeTechnologies) contains a Tn7 transposon to move the DNA encoding theprotein of interest into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.In addition, transfer vectors can include an in-frame fusion with DNAencoding a polypeptide extension or affinity tag as disclosed above.Using techniques known in the art, a transfer vector containing azcyto20-, zcyto21-, zcyto22-, zcyto24- and zcyto25-encoding sequence istransformed into E. coli host cells, and the cells are screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, such as Sf9 cells. Recombinant virus thatexpresses zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 protein issubsequently produced. Recombinant viral stocks are made by methodscommonly used the art.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HighFive™ cells; Invitrogen, Carlsbad, Calif.). See, for example, U.S. Pat.No. 5,300,435. Serum-free media are used to grow and maintain the cells.Suitable media formulations are known in the art and can be obtainedfrom commercial suppliers. The cells are grown up from an inoculationdensity of approximately 2−5×10⁵ cells to a density of 1−2×10⁶ cells, atwhich time a recombinant viral stock is added at a multiplicity ofinfection (MOI) of 0.1 to 10, more typically near 3. Procedures used aregenerally known in the art.

Other higher eukaryotic cells can also be used as hosts, including plantcells and avian cells. The use of Agrobacterium rhizogenes as a vectorfor expressing genes in plant cells has been reviewed by Sinkar et al.,J. Biosci. (Bangalore) 11:47-58, 1987.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 polypeptide in bacteria such as E.coli, the polypeptide may be retained in the cytoplasm, typically asinsoluble granules, or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysed,and the granules are recovered and denatured using, for example,guanidine isothiocyanate or urea. The denatured polypeptide can then berefolded and dimerized by diluting the denaturant, such as by dialysisagainst a solution of urea and a combination of reduced and oxidizedglutathione, followed by dialysis against a buffered saline solution. Inthe latter case, the polypeptide can be recovered from the periplasmicspace in a soluble and functional form by disrupting the cells (by, forexample, sonication or osmotic shock) to release the contents of theperiplasmic space and recovering the protein, thereby obviating the needfor denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors.

It is preferred to purify the polypeptides and proteins of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

Expressed recombinant zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25proteins (including chimeric polypeptides and multimeric proteins) arepurified by conventional protein purification methods, typically by acombination of chromatographic techniques. See, in general, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York, 1994. Proteins comprising apolyhistidine affinity tag (typically about 6 histidine residues) arepurified by affinity chromatography on a nickel chelate resin. See, forexample, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteinscomprising a glu-glu tag can be purified by immunoaffinitychromatography according to conventional procedures. See, for example,Grussenmeyer et al., ibid. Maltose binding protein fusions are purifiedon an amylose column according to methods known in the art.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides can also beprepared through chemical synthesis according to methods known in theart, including exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. See, forexample, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al.,Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co.,Rockford, Ill., 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; andAtherton et al., Solid Phase Peptide Synthesis: A Practical Approach,IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageousfor the preparation of smaller polypeptides.

Using methods known in the art, zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 proteins can be prepared as monomers or multimers; glycosylatedor non-glycosylated; pegylated or non-pegylated; and may or may notinclude an initial methionine amino acid residue.

Target cells for use in zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25activity assays include, without limitation, vascular cells (especiallyendothelial cells and smooth muscle cells), hematopoietic (myeloid andlymphoid) cells, liver cells (including hepatocytes, fenestratedendothelial cells, Kupffer cells, and Ito cells), fibroblasts (includinghuman dermal fibroblasts and lung fibroblasts), fetal lung cells,articular synoviocytes, pericytes, chondrocytes, osteoblasts, andprostate epithelial cells. Endothelial cells and hematopoietic cells arederived from a common ancestral cell, the hemangioblast (Choi et al.,Development 125:725-732, 1998).

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins of the presentinvention are characterized by their activity, that is, modulation ofthe proliferation, differentiation, migration, adhesion, or metabolismof responsive cell types. Biological activity of zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 proteins is assayed using in vitro or invivo assays designed to detect cell proliferation, differentiation,migration or adhesion; or changes in cellular metabolism (e.g.,production of other growth factors or other macromolecules). Manysuitable assays are known in the art, and representative assays aredisclosed herein. Assays using cultured cells are most convenient forscreening, such as for determining the effects of amino acidsubstitutions, deletions, or insertions. However, in view of thecomplexity of developmental processes (e.g., angiogenesis, woundhealing), in vivo assays will generally be employed to confirm andfurther characterize biological activity. Certain in vitro models, suchas the three-dimensional collagen gel matrix model of Pepper et al.(Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficientlycomplex to assay histological effects. Assays can be performed usingexogenously produced proteins, or may be carried out in vivo or in vitrousing cells expressing the polypeptide(s) of interest. Assays can beconducted using zcyto20 proteins alone or in combination with othergrowth factors, such as members of the VEGF family or hematopoieticcytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representativeassays are disclosed below.

Activity of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins canbe measured in vitro using cultured cells or in vivo by administeringmolecules of the claimed invention to an appropriate animal model.Assays measuring cell proliferation or differentiation are well known inthe art. For example, assays measuring proliferation include such assaysas chemosensitivity to neutral red dye (Cavanaugh et al.,Investigational New Drugs 8:347-354, 1990), incorporation ofradiolabelled nucleotides (as disclosed by, e.g., Raines and Ross,Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol. Cell. Biol.8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989),incorporation of 5-bromo-2′-deoxyuridine (BrdU) in the DNA ofproliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179,1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall etal., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res.48:4827-4833, 1988). Differentiation can be assayed using suitableprecursor cells that can be induced to differentiate into a more maturephenotype. Assays measuring differentiation include, for example,measuring cell-surface markers associated with stage-specific expressionof a tissue, enzymatic activity, functional activity or morphologicalchanges (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses,161-171, 1989; all incorporated herein by reference).

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 activity may also bedetected using assays designed to measure zcyto20-, zcyto21-, zcyto22-,zcyto24- and zcyto25-induced production of one or more additional growthfactors or other macromolecules. Preferred such assays include those fordetermining the presence of hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGFα),interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),angiogenin, and other macromolecules produced by the liver. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art. Metalloprotease secretion is measured from treated primaryhuman dermal fibroblasts, synoviocytes and chondrocytes. The relativelevels of collagenase, gelatinase and stromalysin produced in responseto culturing in the presence of a zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 protein is measured using zymogram gels (Loita andStetler-Stevenson, Cancer Biology 1:96-106, 1990). Procollagen/collagensynthesis by dermal fibroblasts and chondrocytes in response to a testprotein is measured using ³H-proline incorporation into nascent secretedcollagen. ³H-labeled collagen is visualized by SDS-PAGE followed byautoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685,1990). Glycosaminoglycan (GAG) secretion from dermal fibroblasts andchondrocytes is measured using a 1,9-dimethylmethylene blue dye bindingassay (Farndale et al., Biochim. Biophys. Acta 883:173-177, 1986).Collagen and GAG assays are also carried out in the presence of IL-1α orTGF-α to examine the ability of zcyto20 protein to modify theestablished responses to these cytokines.

Certain members of the protein family comprising zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 have been shown to increase circulatingmonocyte numbers in vivo. Monocyte activation is important in bothinnate and adaptive immunity. For example, activation of monocytes hasbeen shown to stimulate antigen presentation by several mechanisms.Antigen presentation promotes activation and proliferation of T-cells,both cytotoxic and helper T cells. The maturation and activationdendritic cells also promotes activation of T cells and both innate andadaptive immunity. Increases in activated monocytes and macrophages havealso been shown to increase cytolytic activity. Therefore, zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 will be useful as ananti-infectious agent, enhancing innate, cell-mediated and humoralimmune responses. Increases in ICAM staining in CD14+ monocytes was seensuggesting that zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 play arole in monocyte activation. While data show that family members promotean anti-viral response to virus, bacteria and parasites may also beaffected.

Monocyte activation assays are carried out (1) to look for the abilityof zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins to furtherstimulate monocyte activation, and (2) to examine the ability ofzcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins to modulateattachment-induced or endotoxin-induced monocyte activation (Fuhlbriggeet al., J. Immunol. 138: 3799-3802, 1987). IL-1α and TNFα levelsproduced in response to activation are measured by ELISA (Biosource,Inc. Camarillo, Calif.). Monocyte/macrophage cells, by virtue of CD14(LPS receptor), are exquisitely sensitive to endotoxin, and proteinswith moderate levels of endotoxin-like activity will activate thesecells.

Increased levels of monocytes suggest that zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 may have a direct effect on myeloid progenitor cellsin the bone marrow. Increasing differentiation of myeloid progenitorcells to monocytes is essential in restoring immunocompetency, forexample, after chemotherapy. Thus, administration of zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 to patients receiving chemotherapy couldpromote their recovery and ability to resist infection commonlyassociated with chemotherapy regimens. Thus, methods for expanding thenumbers of monocytes or monocyte progenitor cells by either culturingbone marrow or peripheral blood cells with the molecules of the presentinvention such that there is an increase in the monocyte or monocyteprogenitor cells for achieving this effect in vitro or ex vivo. Thepresent invention also provides for the in vivo administration of themolecules of the present invention to a mammal needing increasedmonocyte or monocyte progenitor cells. Increased monocyte and monocyteprogenitor cells can be measured using methods well known to clinicians,physicians, and other persons skilled the art. Monocyte cells areincluded in the myeloid lineage of hematopoietic cells, so affects onother cells in that lineage would not be unusual. For example, when afactor facilitates the differentiation or proliferation of one type ofcell in the myeloid or lymphoid lineage, this can affect production ofother cells with a common progenitor or stem cell.

Hematopoietic activity of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25proteins can be assayed on various hematopoietic cells in culture.Preferred assays include primary bone marrow colony assays and laterstage lineage-restricted colony assays, which are known in the art(e.g., Holly et al., WIPO Publication WO 95/21920). Marrow cells platedon a suitable semi-solid medium (e.g., 50% methylcellulose containing15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSNantibiotic mix) are incubated in the presence of test polypeptide, thenexamined microscopically for colony formation. Known hematopoieticfactors are used as controls. Mitogenic activity of zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 polypeptides on hematopoietic cell linescan be measured as disclosed above.

Cell migration is assayed essentially as disclosed by Kahler et al.(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). Aprotein is considered to be chemotactic if it induces migration of cellsfrom an area of low protein concentration to an area of high proteinconcentration. A typical assay is performed using modified Boydenchambers with a polystryrene membrane separating the two chambers(Transwell; Corning Costar Corp.). The test sample, diluted in mediumcontaining 1% BSA, is added to the lower chamber of a 24-well platecontaining Transwells. Cells are then placed on the Transwell insertthat has been pretreated with 0.2% gelatin. Cell migration is measuredafter 4 hours of incubation at 37° C. Non-migrating cells are wiped offthe top of the Transwell membrane, and cells attached to the lower faceof the membrane are fixed and stained with 0.1% crystal violet. Stainedcells are then extracted with 10% acetic acid and absorbance is measuredat 600 nm. Migration is then calculated from a standard calibrationcurve. Cell migration can also be measured using the matrigel method ofGrant et al. (“Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997).

Cell adhesion activity is assayed essentially as disclosed by LaFleur etal. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter platesare coated with the test protein, non-specific sites are blocked withBSA, and cells (such as smooth muscle cells, leukocytes, or endothelialcells) are plated at a density of approximately 10⁴-10⁵ cells/well. Thewells are incubated at 37° C. (typically for about 60 minutes), thennon-adherent cells are removed by gentle washing. Adhered cells arequantitated by conventional methods (e.g., by staining with crystalviolet, lysing the cells, and determining the optical density of thelysate). Control wells are coated with a known adhesive protein, such asfibronectin or vitronectin.

The activity of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteinscan be measured with a silicon-based biosensor microphysiometer thatmeasures the extracellular acidification rate or proton excretionassociated with receptor binding and subsequent physiologic cellularresponses. An exemplary such device is the Cytosensor™ Microphysiometermanufactured by Molecular Devices, Sunnyvale, Calif. A variety ofcellular responses, such as cell proliferation, ion transport, energyproduction, inflammatory response, regulatory and receptor activation,and the like, can be measured by this method. See, for example,McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth.Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth. 212:49-59,1998; and Van Liefde et al., Eur. J. Pharmacol. 346:87-95, 1998. Themicrophysiometer can be used for assaying adherent or non-adherenteukaryotic or prokaryotic cells. By measuring extracellularacidification changes in cell media over time, the microphysiometerdirectly measures cellular responses to various stimuli, includingzcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins, their agonists,and antagonists. Preferably, the microphysiometer is used to measureresponses of a zcyto20-, zcyto21-, zcyto22-, zcyto24- andzcyto25-responsive eukaryotic cell, compared to a control eukaryoticcell that does not respond to zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 polypeptide. Zcyto20-, zcyto21-, zcyto22-, zcyto24- andzcyto25-responsive eukaryotic cells comprise cells into which a receptorfor zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 has been transfected,thereby creating a cell that is responsive to zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25, as well as cells naturally responsive to zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25. Differences, measured by achange, for example, an increase or diminution in extracellularacidification, in the response of cells exposed to zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 polypeptide, relative to a control notexposed to zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25, are a directmeasurement of zcyto20-, zcyto21-, zcyto22-, zcyto24- andzcyto25-modulated cellular responses. Moreover, such zcyto20-, zcyto21-,zcyto22-, zcyto24- and zcyto25-modulated responses can be assayed undera variety of stimuli. The present invention thus provides methods ofidentifying agonists and antagonists of zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 proteins, comprising providing cells responsive to azcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptide, culturing afirst portion of the cells in the absence of a test compound, culturinga second portion of the cells in the presence of a test compound, anddetecting a change, for example, an increase or diminution, in acellular response of the second portion of the cells as compared to thefirst portion of the cells. The change in cellular response is shown asa measurable change in extracellular acidification rate. Culturing athird portion of the cells in the presence of a zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 protein and the absence of a test compoundprovides a positive control for the zcyto20-, zcyto21-, zcyto22-,zcyto24- and zcyto25-responsive cells and a control to compare theagonist activity of a test compound with that of the zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 polypeptide. Antagonists of zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 can be identified by exposing thecells to zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 protein in thepresence and absence of the test compound, whereby a reduction inzcyto20-, zcyto21-, zcyto22-, zcyto24- and zcyto25-stimulated activityis indicative of antagonist activity in the test compound.

Expression of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polynucleotides in animals provides models for further study of thebiological effects of overproduction or inhibition of protein activityin vivo. Zcyto20-, zcyto21-, zcyto22-, zcyto24- and zcyto25-encodingpolynucleotides and antisense polynucleotides can be introduced intotest animals, such as mice, using viral vectors or naked DNA, ortransgenic animals can be produced.

One in vivo approach for assaying proteins of the present inventionutilizes viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene is deleted from the viral vector, and the virus willnot replicate unless the E1 gene is provided by the host cell (e.g., thehuman 293 cell line). When intravenously administered to intact animals,adenovirus primarily targets the liver. If the adenoviral deliverysystem has an E1 gene deletion, the virus cannot replicate in the hostcells. However, the host's tissue (e.g., liver) will express and process(and, if a signal sequence is present, secrete) the heterologousprotein. Secreted proteins will enter the circulation in the highlyvascularized liver, and effects on the infected animal can bedetermined.

An alternative method of gene delivery comprises removing cells from thebody and introducing a vector into the cells as a naked DNA plasmid. Thetransformed cells are then re-implanted in the body. Naked DNA vectorsare introduced into host cells by methods known in the art, includingtransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem.263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967, 1992; andJohnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

Transgenic mice, engineered to express a zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 gene, and mice that exhibit a complete absence ofzcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 gene function, referredto as “knockout mice” (Snouwaert et al., Science 257:1083, 1992), canalso be generated (Lowell et al., Nature 366:740-742, 1993). These micecan be employed to study the zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 gene and the protein encoded thereby in an in vivo system.Transgenic mice are particularly useful for investigating the role ofzcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins in earlydevelopment in that they allow the identification of developmentalabnormalities or blocks resulting from the over- or underexpression of aspecific factor. See also, Maisonpierre et al., Science 277:55-60, 1997and Hanahan, Science 277:48-50, 1997. Preferred promoters for transgenicexpression include promoters from metallothionein and albumin genes.

A loss of normal inhibitory control of muscle contraction has beenassociated with damage or perturbation of selected gamma-aminobutyricacid-secreting neurons. For example, Stiff Man Syndrome exhibitremarkable stiffness of musculature, believed to be mediated throughinterference of the functioning of their gamma-aminobutyric acid (GABA)producing neurons. Other related neuromuscular disorders includemyotonia, metabolic myopathies, Isaac's syndrome, dystonia, and tetanicspasms (Valldeoriola, J. Neurol 246:423-431, 1999).

Similarly, direct measurement of zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 polypeptide, or its loss of expression in a tissue can bedetermined in a tissue or cells as they undergo tumor progression.Increases in invasiveness and motility of cells, or the gain or loss ofexpression of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 in apre-cancerous or cancerous condition, in comparison to normal tissue,can serve as a diagnostic for transformation, invasion and metastasis intumor progression. As such, knowledge of a tumor's stage of progressionor metastasis will aid the physician in choosing the most propertherapy, or aggressiveness of treatment, for a given individual cancerpatient. Methods of measuring gain and loss of expression (of eithermRNA or protein) are well known in the art and described herein and canbe applied to zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 expression.For example, appearance or disappearance of polypeptides that regulatecell motility can be used to aid diagnosis and prognosis of prostatecancer (Banyard, J. and Zetter, B. R., Cancer and Metast. Rev.17:449-458, 1999). As an effector of cell motility, or as aliver-specific marker, zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25gain or loss of expression may serve as a diagnostic for brain and othercancers. Moreover, analogous to the prostate specific antigen (PSA),increased levels of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polypeptides, or anti-zcyto20, anti-zcyto21, anti-zcyto22, anti-zcyto24and anti-zcyto25 antibodies in a patient, relative to a normal controlcan be indicative of brain and other cancers (See, e.g., Mulders, T M T,et al., Eur. J. Surgical Oncol. 16:37-41, 1990). Strong zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 expression in tissue not normallyfound to express the polynucleotides would serve as a diagnostic of anabnormality in the cell or tissue type, of invasion or metastasis ofcancerous liver tissue into non-liver tissue, and could aid a physicianin directing further testing or investigation, or aid in directingtherapy.

In addition, zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polynucleotide probes, anti-zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 antibodies, and detection the presence of zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 polypeptides in tissue can be used toassess whether brain or other tissue found to normally express zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 is present, for example, aftersurgery involving the excision of a diseased or cancerous liver orneuronal tissue. As such, the polynucleotides, polypeptides, andantibodies of the present invention can be used as an aid to determinewhether all tissue is excised after surgery, for example, after surgeryfor brain and other cancers. In such instances, it is especiallyimportant to remove all potentially diseased tissue to maximize recoveryfrom the cancer, and to minimize recurrence. Preferred embodimentsinclude fluorescent, radiolabeled, or calorimetrically labeledanti-zcyto20 antibodies and zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 polypeptide binding partners, that can be used histologically orin situ.

Moreover, the activity and effect of zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 on tumor progression and metastasis can be measured in vivo.Several syngeneic mouse models have been developed to study theinfluence of polypeptides, compounds or other treatments on tumorprogression. In these models, tumor cells passaged in culture areimplanted into mice of the same strain as the tumor donor. The cellswill develop into tumors having similar characteristics in the recipientmice, and metastasis will also occur in some of the models. Appropriatetumor models for our studies include the Lewis lung carcinoma (ATCC No.CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. Theseare both commonly used tumor lines, syngeneic to the C57BL6 mouse, thatare readily cultured and manipulated in vitro. Tumors resulting fromimplantation of either of these cell lines are capable of metastasis tothe lung in C57BL6 mice. The Lewis lung carcinoma model has recentlybeen used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328, 1994). C57BL6/J mice are treated with anexperimental agent either through daily injection of recombinantprotein, agonist or antagonist or a one-time injection of recombinantadenovirus. Three days following this treatment, 10⁵ to 10⁶ cells areimplanted under the dorsal skin. Alternatively, the cells themselves maybe infected with recombinant adenovirus, such as one expressing zcyto20,zcyto21, zcyto22, zcyto24 or zcyto25, before implantation so that theprotein is synthesized at the tumor site or intracellularly, rather thansystemically. The mice normally develop visible tumors within 5 days.The tumors are allowed to grow for a period of up to 3 weeks, duringwhich time they may reach a size of 1500-1800 mm³ in the control treatedgroup. Tumor size and body weight are carefully monitored throughout theexperiment. At the time of sacrifice, the tumor is removed and weighedalong with the lungs and the liver. The lung weight has been shown tocorrelate well with metastatic tumor burden. As an additional measure,lung surface metastases are counted. The resected tumor, lungs and liverare prepared for histopathological examination, immunohistochemistry,and in situ hybridization, using methods known in the art and describedherein. The influence of the expressed polypeptide in question, e.g.,zcyto20, zcyto21, zcyto22, zcyto24 or zcyto25, on the ability of thetumor to recruit vasculature and undergo metastasis can thus beassessed. In addition, aside from using adenovirus, the implanted cellscan be transiently transfected with zcyto20, zcyto21, zcyto22, zcyto24or zcyto25. Use of stable zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25transfectants as well as use of induceable promoters to activatezcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 expression in vivo areknown in the art and can be used in this system to assess zcyto20induction of metastasis. Moreover, purified zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 or zcyto20-, zcyto21-, zcyto22-, zcyto24- andzcyto25-conditioned media can be directly injected in to this mousemodel, and hence be used in this system. For general reference see,O'Reilly M S, et al. Cell 79:315-328, 1994; and Rusciano D, et al.Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

Antisense methodology can be used to inhibit zcyto20 gene transcriptionto examine the effects of such inhibition in vivo. Polynucleotides thatare complementary to a segment of a zcyto20-, zcyto21-, zcyto22-,zcyto24- and zcyto25-encoding polynucleotide (e.g., a polynucleotide asset forth in SEQ ID NO:1) are designed to bind to zcyto20-, zcyto21-,zcyto22-, zcyto24- and zcyto25-encoding mRNA and to inhibit translationof such mRNA. Such antisense oligonucleotides can also be used toinhibit expression of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polypeptide-encoding genes in cell culture.

Most cytokines as well as other proteins produced by activatedlymphocytes play an important biological role in cell differentiation,activation, recruitment and homeostasis of cells throughout the body.Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 and inhibitors of theiractivity are expected to have a variety of therapeutic applications.These therapeutic applications include treatment of diseases whichrequire immune regulation, including autoimmune diseases such asrheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemiclupus erythematosis, and diabetes. Zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 may be important in the regulation of inflammation, andtherefore would be useful in treating rheumatoid arthritis, asthma andsepsis. There may be a role of zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 in mediating tumorgenesis, whereby a zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 antagonist would be useful in the treatment ofcancer. Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 may be useful inmodulating the immune system, whereby zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 and zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25antagonists may be used for reducing graft rejection, preventinggraft-vs-host disease, boosting immunity to infectious diseases,treating immunocompromised patients (e.g., HIV⁺ patients), or inimproving vaccines.

Members of the protein family of the present invention have been shownto have an antiviral effect that is similar to interferon-α. Interferonhas been approved in the United States for treatment of autoimmunediseases, condyloma acuminatum, chronic hepatitis C, bladder carcinoma,cervical carcinoma, laryngeal papillomatosis, fungoides mycosis, chronichepatitis B, Kaposi's sarcoma in patients infected with humanimmunodeficiency virus, malignant melanoma, hairy cell leukemia, andmultiple sclerosis. In addition, zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 may be used to treat forms of arteriosclerosis, such asatherosclerosis, by inhibiting cell proliferation. Accordingly, thepresent invention contemplates the use of proteins, polypeptides, andpeptides having zcyto20 activity to treat such conditions, as well as totreat retinopathy. The present invention also contemplates the use ofproteins, polypeptides, and peptides having zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 activity to treat lymphoproliferative disorders,including B-cell lymphomas, chronic lymphocytic leukemia, acutelymphocytic leukemia, Non-Hodkin's lymphomas, multiple myeloma, acutemyelocytic leukemia, chronic myelocytic leukemia.

Interferons have also been shown to induce the expression of antigens bycultured cells (see, for example, Auth et al., Hepatology 18:546 (1993),Guadagni et al., Int. J. Biol. Markers 9:53 (1994), Girolomoni et al.,Eur. J. Immunol. 25:2163 (1995), and Maciejewski et al., Blood 85:3183(1995). This activity enhances the ability to identify new tumorassociated antigens in vitro. Moreover, the ability of interferons toaugment the level of expression of human tumor antigens indicates thatinterferons can be useful in an adjuvant setting for immunotherapy orenhance immunoscintigraphy using anti-tumor antigen antibodies (Guadagniet al., Cancer Immunol. Immunother. 26:222 (1988); Guadagni et al., Int.J. Biol. Markers 9:53 (1994)). Thus, the present invention includes theuse of proteins, polypeptides and peptides having zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 activity as an adjuvant for immunotherapyor to improve immunoscintigraphy using anti-tumor antigen antibodies.

Methods for detection and diagnosis of viral infections are well knownto those skilled in the art. The exact method used for measuring areduction in virus in response to administration of molecules of thepresent invention will be dependent upon the patient, type of viralinfection, and the like. For example, methods include, but are notlimited to, measuring changes in CD4 cell counts, serologic tests,measuring the DNA of the virus and RNA of the virus by conventional andreal-time quantitative polymerase chain reaction assays, viral inducedantibody levels, immunofluorescence and enzyme-linked immunosorbantassays, cytopathic effects, and histology.

Moreover, zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 may bind CD4 oranother leukocyte receptor and exhibit antiviral effects, for example,against human immunodeficiency virus (HIV) or human T-cell lymphotropicvirus (HTLV). Alternatively, zcyto20 polypeptide may compete for a viralreceptor or co-receptor to block viral infection. Zcyto20 may be givenparentally to prevent viral infection or to reduce ongoing viralreplication and re-infection (Gayowski, T. et al., Transplantation64:422-426, 1997). Thus, zcyto20 may be used as an antiviraltherapeutic, for example, for viral leukemias (HTLV), AIDS (HIV), orgastrointestinal viral infections caused by, for example, rotavirus,calicivirus (e.g., Norwalk Agent) and certain strains of pathogenicadenovirus, Hepatitis B and C.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can also be used to treatmyocarditis, a disorder that arises when the heart is involved in aninflammatory process. The infiltration of lymphocytes and myocytolysisis thought to result after infection by virus, bacteria, fungi orparasites (see, for example, Brodison et al., J. Infection 37:99(1998)). Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can be injectedintravenously or subcutaneously to treat infections associated withmyocarditis. Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can also beadministered intravenously as an immunoregulatory cytokine in thetreatment of autoimmune myocarditis. Interferon dosages can beextrapolated using a autoimmune model of myocarditis in the A/J mouse(Donermeyer, et al., J. Exp. Med. 182:1291 (1995)).

Exogenous administration of interferon-τ in sheep increases thepregnancy rate (Aggarwal, Human Cytokines III, (Blackwell Science1997)). As described herein, Zcyto20mRNA is expressed in placenta.Accordingly, the present invention includes the use of zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25, such as the disclosed human zcyto20,zcyto21, and zcyto22, to promote and protect growth of the fetus. As anillustration, zcyto20, zcyto21, and zcyto22 can be used to protect adeveloping fetus from viral infection (e.g., human immunodeficiencyvirus, human papilloma virus, and the like). In addition, zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 can be used to promote in vitrofertilization.

Recent reports have highlighted the role of type I interferons in theprevention of viral-induced diabetes by inducing a strong antiviralstate in pancreatic beta cells early during viral infection (Flodstroemet al., Nature Immunology 3, 373-382 (2002)). This prevents the loss ofbeta cells due to viral-induced cell death and autoimmunity thataccompanies it. Zcyto20, 21 and 22 also induce an antiviral state incells that express their receptor, zcytor19. Zcytor19 is highlyexpressed in pancreatic tissue and therefore zcyto20-22 may play a rolein prevention of viral-induced diabetes due to beta cell death. Inaddition, the role of type I interferons in prevention of viral-induceddiabetes may be extended to other viral-induced autoimmune diseases andtherefore, zcyto20-22 may also play a role in prevention of otherdiseases such as muscular sclerosis, lupus, and viral-induced autoimmunediseases in tissues that express the zcyto20-22 receptor, zcytor19.

Chronic systemic expression of type I interferons has also beenassociated with the pathogenesis of type I diabetes. Given thesimilarity of type I interferons to zcyto20-22 in regards to biologicalactivity and gene induction, chronic systemic expression of zcyto20, 21,or 22 might also play a role in the pathogenesis of type I diabetes.Therefore, an inhibitor of zcyto20-22 activity in the pancreas might bebeneficial in prevention of type I diabetes.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides can beadministered alone or in combination with other vasculogenic orangiogenic agents, including VEGF. When using zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 in combination with an additional agent, the twocompounds can be administered simultaneously or sequentially asappropriate for the specific condition being treated.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 will be useful intreating tumorgenesis, and therefore would be useful in the treatment ofcancer. A zcyto20 inhibition of anti-IgM stimulated normal B-cells and asimilar effect is observed in B-cell tumor lines suggest that there maybe therapeutic benefit in treating patients with the zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 in order to induce the B cell tumor cellsinto a less proliferative state. The ligand could be administered incombination with other agents already in use including both conventionalchemotherapeutic agents as well as immune modulators such as interferonalpha. Alpha/beta interferons have been shown to be effective intreating some leukemias and animal disease models, and the growthinhibitory effects of interferon-alpha and zcyto20 may be additive forB-cell tumor-derived cell lines.

The present invention provides a method of reducing proliferation of aneoplastic B or T cells comprising administering to a mammal with a B orT cell neoplasm an amount of a composition of zcyto20 sufficient toreduce proliferation of the neoplastic B or T cells. Zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 stimulation of lytic NK cells from marrowprogenitors and the proliferation of T-cells following activation of theantigen receptors would enhance treatment for patients receivingallogenic marrow transplants, and therefore, zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 will enhance the generation of anti-tumor responses,with or without the infusion of donor lymphocytes.

In another aspect, the present invention provides a method of reducingproliferation of a neoplastic B or T cells comprising administering to amammal with a B or T cell neoplasm an amount of a composition of zcyto20antagonist sufficient to reducing proliferation of the neoplastic B or Tcells. Furthermore, the zcyto20 antagonist can be a ligand/toxin fusionprotein.

A zcyto20-, zcyto21-, zcyto22-, zcyto24- and zcyto25-saporin fusiontoxin may be employed against a similar set of leukemias and lymphomas,extending the range of leukemias that can be treated with zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25. Fusion toxin mediated activationof the zcyto20 receptor provides two independent means to inhibit thegrowth of the target cells, the first being identical to the effectsseen by the ligand alone, and the second due to delivery of the toxinthrough receptor internalization.

For pharmaceutical use, zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25proteins are formulated for topical or parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.In general, pharmaceutical formulations will include a zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 polypeptide in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water, or the like. Formulations may further include one ormore excipients, preservatives, solubilizers, buffering agents, albuminto prevent protein loss on vial surfaces, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington:The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 will preferably be used in a concentration of about 10 to 100μg/ml of total volume, although concentrations in the range of 1 ng/mlto 1000 μg/ml may be used. For topical application, such as for thepromotion of wound healing, the protein will be applied in the range of0.1-10 μg/cm² of wound area, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.Dosing is daily or intermittently over the period of treatment.Intravenous administration will be by bolus injection or infusion over atypical period of one to several hours. Sustained release formulationscan also be employed. In general, a therapeutically effective amount ofzcyto20 is an amount sufficient to produce a clinically significantchange in the treated condition, such as a clinically significant changein hematopoietic or immune function, a significant reduction inmorbidity, or a significantly increased histological score.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins, agonists, andantagonists are useful for modulating the expansion, proliferation,activation, differentiation, migration, or metabolism of responsive celltypes, which include both primary cells and cultured cell lines. Ofparticular interest in this regard are hematopoietic cells, mesenchymalcells (including stem cells and mature myeloid and lymphoid cells),endothelial cells, smooth muscle cells, fibroblasts, hepatocytes, neuralcells and embryonic stem cells. Zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 polypeptides are added to tissue culture media for these celltypes at a concentration of about 10 pg/ml to about 100 ng/ml. Thoseskilled in the art will recognize that zcyto20 proteins can beadvantageously combined with other growth factors in culture media.

Within the laboratory research field, zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 proteins can also be used as molecular weight standards oras reagents in assays for determining circulating levels of the protein,such as in the diagnosis of disorders characterized by over- orunder-production of zcyto20 protein or in the analysis of cellphenotype.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins can also be usedto identify inhibitors of their activity. Test compounds are added tothe assays disclosed above to identify compounds that inhibit theactivity of zcyto20 protein. In addition to those assays disclosedabove, samples can be tested for inhibition of zcyto20 activity within avariety of assays designed to measure receptor binding or thestimulation/inhibition of zcyto20-, zcyto21-, zcyto22-, zcyto24- andzcyto25-dependent cellular responses. For example, zcyto20-, zcyto21-,zcyto22-, zcyto24- and zcyto25-responsive cell lines can be transfectedwith a reporter gene construct that is responsive to a zcyto20-,zcyto21-, zcyto22-, zcyto24- and zcyto25-stimulated cellular pathway.Reporter gene constructs of this type are known in the art, and willgenerally comprise a zcyto20-, zcyto21-, zcyto22-, zcyto24- andzcyto25-activated serum response element (SRE) operably linked to a geneencoding an assayable protein, such as luciferase. Candidate compounds,solutions, mixtures or extracts are tested for the ability to inhibitthe activity of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 on thetarget cells as evidenced by a decrease in zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 stimulation of reporter gene expression. Assays ofthis type will detect compounds that directly block zcyto20, zcyto21,zcyto22, zcyto24 and zcyto25 binding to cell-surface receptors, as wellas compounds that block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 binding to receptor using zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 tagged with a detectable label (e.g., ¹²⁵I, biotin,horseradish peroxidase, FITC, and the like). Within assays of this type,the ability of a test sample to inhibit the binding of labeled zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 to the receptor is indicative ofinhibitory activity, which can be confirmed through secondary assays.Receptors used within binding assays may be cellular receptors orisolated, immobilized receptors.

As used herein, the term “antibodies” includes polyclonal antibodies,monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)₂and Fab fragments, single chain antibodies, and the like, includinggenetically engineered antibodies. Non-human antibodies may be humanizedby grafting non-human CDRs onto human framework and constant regions, orby incorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. One skilled in the art can generatehumanized antibodies with specific and different constant domains (i.e.,different Ig subclasses) to facilitate or inhibit various immunefunctions associated with particular antibody constant domains.Antibodies are defined to be specifically binding if they bind to azcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptide or proteinwith an affinity at least 10-fold greater than the binding affinity tocontrol (non-zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25) polypeptideor protein. The affinity of a monoclonal antibody can be readilydetermined by one of ordinary skill in the art (see, for example,Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982, which is incorporated herein by reference). Ofparticular interest are generating antibodies to hydrophilic antigenicsites which include, for example, amino acid residues 169 (Glu) to 174(Glu) of SEQ ID NO: 2, amino acid residues 54 (Lys) to 59 (Ala) of SEQID NO: 2, amino acid residues 53 (Phe) to 58 (Asp) of SEQ ID NO: 2,amino acid residues 168 (Gln) to 173 (Lys) of SEQ ID NO: 2, and aminoacid residues 154 (Pro) to 159 (Arg) of SEQ ID NO: 2. For example, inzcyto22, hydrophilic regions include amino acid residues 169 (Glu) to174 (Glu) of SEQ ID NO: 7, amino acid residues 54 (Lys) to 59 (Ala) ofSEQ ID NO: 7, amino acid residues 53 (Phe) to 58 (Asp) of SEQ ID NO: 7,amino acid residues 168 (Gln) to 173 (Lys) of SEQ ID NO: 7, and aminoacid residues 154 (Pro) to 159 (Arg) of SEQ ID NO: 7 would be useful. Aswould be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from a variety of warm-blooded animals suchas horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.The immunogenicity of a zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polypeptide may be increased through the use of an adjuvant such as alum(aluminum hydroxide) or Freund's complete or incomplete adjuvant.Polypeptides useful for immunization also include fusion polypeptides,such as fusions of a zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polypeptide or a portion thereof with an immunoglobulin polypeptide orwith maltose binding protein. The polypeptide immunogen may be afull-length molecule or a portion thereof. If the polypeptide portion is“hapten-like”, such portion may be advantageously joined or linked to amacromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA) or tetanus toxoid) for immunization.

Alternative techniques for generating or selecting antibodies include invitro exposure of lymphocytes to zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 polypeptides, and selection of antibody display libraries inphage or similar vectors (e.g., through the use of immobilized orlabeled zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptide).Human antibodies can be produced in transgenic, non-human animals thathave been engineered to contain human immunoglobulin genes as disclosedin WIPO Publication WO 98/24893. It is preferred that the endogenousimmunoglobulin genes in these animals be inactivated or eliminated, suchas by homologous recombination.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 polypeptides. Exemplary assays are described indetail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), ColdSpring Harbor Laboratory Press, 1988. Representative examples of suchassays include: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA),dot blot assays, Western blot assays, inhibition or competition assays,and sandwich assays.

Antibodies to zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 may be usedfor affinity purification of the protein, within diagnostic assays fordetermining circulating levels of the protein; for detecting orquantitating soluble zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25polypeptide as a marker of underlying pathology or disease; forimmunolocalization within whole animals or tissue sections, includingimmunodiagnostic applications; for immunohistochemistry; and asantagonists to block protein activity in vitro and in vivo. Antibodiesto zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 may also be used fortagging cells that express zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25; for affinity purification of zcyto20, zcyto21, zcyto22, zcyto24and zcyto25 polypeptides and proteins; in analytical methods employingFACS; for screening expression libraries; and for generatinganti-idiotypic antibodies. Antibodies can be linked to other compounds,including therapeutic and diagnostic agents, using known methods toprovide for targeting of those compounds to cells expressing receptorsfor zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25. For certainapplications, including in vitro and in vivo diagnostic uses, it isadvantageous to employ labeled antibodies. Suitable direct tags orlabels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies of the present invention may also be directlyor indirectly conjugated to drugs, toxins, radionuclides and the like,and these conjugates used for in vivo diagnostic or therapeuticapplications (e.g., inhibition of cell proliferation). See, in general,Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.

Polypeptides and proteins of the present invention can be used toidentify and isolate receptors. Zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 receptors may be involved in growth regulation in the liver,blood vessel formation, and other developmental processes. For example,zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins and polypeptidescan be immobilized on a column, and membrane preparations run over thecolumn (as generally disclosed in Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp. 195-202). Proteins and polypeptides can also be radiolabeled(Methods Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Academic Press, San Diego, 1990, 721-737) orphotoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514,1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and usedto tag specific cell-surface proteins. In a similar manner, radiolabeledzcyto20 proteins and polypeptides can be used to clone the cognatereceptor in binding assays using cells transfected with an expressioncDNA library.

Zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides can also beused to teach analytical skills such as mass spectrometry, circulardichroism, to determine conformation, especially of the four alphahelices, x-ray crystallography to determine the three-dimensionalstructure in atomic detail, nuclear magnetic resonance spectroscopy toreveal the structure of proteins in solution. For example, a kitcontaining the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can begiven to the student to analyze. Since the amino acid sequence would beknown by the instructor, the protein can be given to the student as atest to determine the skills or develop the skills of the student, theinstructor would then know whether or not the student has correctlyanalyzed the polypeptide. Since every polypeptide is unique, theeducational utility of zcyto20 would be unique unto itself.

The antibodies which bind specifically to zcyto20, zcyto21, zcyto22,zcyto24 and zcyto25 can be used as a teaching aid to instruct studentshow to prepare affinity chromatography columns to purify zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25, cloning and sequencing thepolynucleotide that encodes an antibody and thus as a practicum forteaching a student how to design humanized antibodies. The zcyto20,zcyto21, zcyto22, zcyto24 and zcyto25 gene, polypeptide, or antibodywould then be packaged by reagent companies and sold to educationalinstitutions so that the students gain skill in art of molecularbiology. Because each gene and protein is unique, each gene and proteincreates unique challenges and learning experiences for students in a labpracticum. Such educational kits containing the ZCYTO20 gene,polypeptide, or antibody are considered within the scope of the presentinvention.

In summary, the present invention provides an isolated polypeptide thathas at least 80% or 95% or 100% identity to a polypeptide selected fromthe group consisting: (a) a polypeptide comprising an amino acidsequence as shown in SEQ ID NO:2 from amino acid residue 22 to aminoacid residue 205; (b) a polypeptide comprising an amino acid sequence asshown in SEQ ID NO: 7 from amino acid residue 22 to amino acid residue205; (c) a polypeptide comprising an amino acid sequence as shown in SEQID NO: 9 from amino acid residue 29 to amino acid residue 202; and (d) apolypeptide comprising an amino acid sequence as shown in SEQ ID NO: 11from amino acid residue 29 to amino acid residue 202.

In another embodiment, the isolated polypeptide binds a receptor asshown in SEQ ID NOS: 24, 27 or 29 as a monomeric or homodimeric receptoror SEQ ID NOS: 24, 27 or 29 in combination with SEQ ID NO:41 as aheterodimeric receptor.

In another aspect, the present invention includes an isolatedpolypeptide comprising an amino acid sequence as shown in SEQ ID NO:2from amino acid residue 22 to amino acid residue 205 or as shown in SEQID NO:2 from amino acid residue 1 to amino acid residue 205.

In another aspect, the present invention includes an isolatedpolypeptide comprising an amino acid sequence as shown in SEQ ID NO:7from amino acid residue 22 to amino acid residue 205 or as shown in SEQID NO:7 from amino acid residue 1 to amino acid residue 205.

In another aspect, the present invention includes an isolatedpolypeptide comprising an amino acid sequence as shown in SEQ ID NO: 9from amino acid residue 29 to amino acid residue 202 or as shown in SEQID NO:9 from amino acid residue 1 to amino acid residue 202.

In another aspect, the present invention includes an isolatedpolypeptide comprising an amino acid sequence as shown in SEQ ID NO: 11from amino acid residue 29 to amino acid residue 202 or as shown in SEQID NO: 11 from amino acid residue 1 to amino acid residue 202.

In another aspect, the present invention includes an isolatedpolypeptide that is at least 14 contiguous amino acids of SEQ ID NO:2from amino acid residue 22 to amino acid residue 205 or as shown in SEQID NO:2 from amino acid residue 1 to amino acid residue 205, whereinsaid polypeptide stimulates an antigenic response in a mammal.

In another aspect, the present invention includes an isolatedpolypeptide that is at least 14 contiguous amino acids of SEQ ID NO:7from amino acid residue 22 to amino acid residue 205 or as shown in SEQID NO:7 from amino acid residue 1 to amino acid residue 205, whereinsaid polypeptide stimulates an antigenic response in a mammal.

In another aspect, the present invention includes an isolatedpolypeptide that is at least 14 contiguous amino acids of SEQ ID NO: 9from amino acid residue 29 to amino acid residue 202 or as shown in SEQID NO:9 from amino acid residue 1 to amino acid residue 202, whereinsaid polypeptide stimulates an antigenic response in a mammal.

In another aspect, the present includes an isolated polypeptide that isat least 14 contiguous amino acids of SEQ ID NO: 11 from amino acidresidue 29 to amino acid residue 202 or as shown in SEQ ID NO: 11 fromamino acid residue 1 to amino acid residue 202, wherein said polypeptidestimulates an antigenic response in a mammal.

The present invention includes pharmaceutical compositions comprisingthe polypeptides described herein, in pharmaceutically acceptablevehicles.

The present invention also includes fusion proteins comprising thepolypeptides described herein.

In other aspects, the present invention includes an isolatedpolynucleotide that encodes a polypeptide, wherein the nucleic acidmolecule is selected from the group consisting of: (a) a polynucleotidecomprising the nucleotide sequence of SEQ ID NO:3, (b) a polynucleotidethat remains hybridized following stringent wash conditions to apolynucleotide consisting of the nucleotide sequence of nucleotides 64to 618 of SEQ ID NO:1, or the complement of the nucleotide sequence ofnucleotides 64 to 618 of SEQ ID NO:1.

In another embodiment, the isolated polynucleotide that encodes apolypeptide, wherein the nucleic acid molecule is selected from thegroup consisting of: (a) a polynucleotide comprising the nucleotidesequence of SEQ ID NO:36, (b) a polynucleotide that remains hybridizedfollowing stringent wash conditions to a polynucleotide consisting ofthe nucleotide sequence of nucleotides 64 to 618 of SEQ ID NO:6, or thecomplement of the nucleotide sequence of nucleotides 64 to 618 of SEQ IDNO:6.

In another aspect, the present invention includes an isolatedpolynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:1from nucleotide 64 to nucleotide 618 or as shown in SEQ ID NO:1 fromnucleotide 1 to nucleotide 618.

In another embodiment, the present invention includes an isolatedpolynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:6from nucleotide 64 to nucleotide 618 or as shown in SEQ ID NO:6 fromnucleotide 1 to nucleotide 618.

In another embodiment, the present invention includes an isolatedpolynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:8from nucleotide 67 to nucleotide 606 or as shown in SEQ ID NO:1 fromnucleotide 1 to nucleotide 606.

In another embodiment, the present invention includes an isolatedpolynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:10from nucleotide 67 to nucleotide 606 or as shown in SEQ ID NO:10 fromnucleotide 1 to nucleotide 606.

The present invention provides expression vectors, comprising theisolated nucleic acid molecules described herein with a transcriptionpromoter, and a transcription terminator, wherein the promoter isoperably linked with the nucleic acid molecule, and wherein the nucleicacid molecule is operably linked with the transcription terminator.

The present invention includes provides recombinant host cellscomprising the expression vector described herein, wherein the host cellis selected from the group consisting of bacterium, yeast cell, fungalcell, insect cell, mammalian cell, and plant cell.

In another aspect, the present invention provides a method of producinga polypeptide, the method comprising the step of culturing recombinanthost cells that comprise the expression vectors described herein, andthat produce the polypeptides.

The present invention provides an antibody or antibody fragment thatspecifically binds with the polypeptides described herein.

In another aspect, the present invention provides a method for expansionof monocytic cells or monocyte cell progenitors comprising culturingbone marrow or peripheral blood cells with a composition comprising anamount of the polypeptides described herein sufficient to produce anincrease in the number of monocytic cells or monocyte cell progenitorsin the bone marrow or peripheral blood cells as compared to bone marrowor peripheral blood cells cultured in the absence of administeredpolypeptide.

The present invention also provides a method of stimulating an immuneresponse in a mammal exposed to an antigen or pathogen comprising: (1)determining a level of an antigen- or pathogen-specific antibody; (2)administering a composition comprising the polypeptides described hereinin an acceptable pharmaceutical vehicle; (3) determining a postadministration level of antigen- or pathogen-specific antibody; (4)comparing the level of antibody in step (1) to the level of antibody instep (3), wherein an increase in antibody level is indicative ofstimulating an immune response.

In other aspects, the present invention provides a method of producingan anti-viral response in a mammal comprising administering to a mammalwith a viral infection an amount of a composition of the polypeptidesdescribed herein sufficient to show a reduction in virus.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which is provided by wayof illustration and is not intended to be limiting of the presentinvention.

EXAMPLES Example 1 Mammalian Expression Plasmids

An expression plasmid containing a polynucleotide encoding zcyto20,zcyto21, zcyto22, zcyto24 or zcyto25 can be constructed via homologousrecombination. A fragment of cDNA, for example zcyto20 cDNA, is isolatedby PCR using the polynucleotide sequence of SEQ ID NO: 1 with flankingregions at the 5′ and 3′ ends corresponding to the vector sequencesflanking the zcyto20 insertion point. The primers ZC40923 and ZC40927are shown in SEQ ID NOS: 12 and 13, respectively.

The PCR reaction mixture is run on a 1% agarose gel and a bandcorresponding to the size of the insert is gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.). Plasmid pZMP21is a mammalian expression vector containing an expression cassettehaving the MPSV promoter, multiple restriction sites for insertion ofcoding sequences, a stop codon, an E. coli origin of replication; amammalian selectable marker expression unit comprising an SV40 promoter,enhancer and origin of replication, a DHFR gene, and the SV40terminator; and URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae. It is constructed from pZP9 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, under Accession No. 98668) with the yeastgenetic elements taken from pRS316 (deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under Accession No. 77145), an internal ribosome entry site(IRES) element from poliovirus, and the extracellular domain of CD8truncated at the C-terminal end of the transmembrane domain. PlasmidpZMP21 was digested with BgIII, and used for recombination with the PCRinsert.

One hundred microliters of competent yeast (S. cerevisiae) cells areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed using power supply (BioRad Laboratories,Hercules, Calif.) settings of 0.75 kV (5 kV/cm), cc ohms, 25 PF. To eachcuvette is added 600 μl of 1.2 M sorbitol, and the yeast is plated intwo 300-μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura⁺ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid-washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, and spun for 5minutes in an Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA is precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet is resuspended in10 μl H₂O.

Transformation of electrocompetent E. coli host cells (Electromax DH10B™cells; obtained from Life Technologies, Inc., Gaithersburg, Md.) is donewith 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells areelectropulsed at 1.7 kV, 25 pF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for zcyto20are identified by restriction digest to verify the presence of thezcyto20 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated zcyto20-CEE/pZMP21.

Plasmids containing zcyto21, zcyto22, zcyto24 or zcyto25 are preparedsimilarly, using nucleotide-specific primers

Example 2 Expression in Chinese Hamster Ovary Cells

CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,1986) are plated in 10-cm tissue culture dishes and allowed to grow toapproximately 50% to 70% confluency overnight at 37° C., 5% CO₂, inHam's F12/FBS media (Ham's F12 medium (Life Technologies), 5% fetalbovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRH Biosciences,Lenexa, Kans.), 1% sodium pyruvate (Life Technologies)). The cells arethen transfected with a plasmid containing zcyto20, zcyto21, zcyto22,zcyto24 or zcyto25, i.e. zcyto20/pZMP6, by liposome-mediatedtransfection using a 3:1 (w/w) liposome formulation of the polycationiclipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filtered water (Lipofectamine™ Reagent, Life Technologies), inserum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Zcyto20/pZMP6 is diluted into 15-ml tubes to a total final volume of 640μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl of SFmedium. The resulting mixture is added to the DNA mixture and allowed toincubate approximately 30 minutes at room temperature. Five ml of SFmedia is added to the DNA:Lipofectamine™ mixture. The cells are rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine mixtureis added. The cells are incubated at 37° C. for five hours, then 6.4 mlof Ham's F12/10% FBS, 1% PSN media is added to each plate. The platesare incubated at 37° C. overnight, and the DNA:Lipofectamine mixture isreplaced with fresh 5% FBS/Ham's media the next day. On day 3post-transfection, the cells are split into T-175 flasks in growthmedium. On day 7 postransfection, the cells are stained withFITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, Calif.)followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec,Auburn, Calif.). The CD8-positive cells are separated using commerciallyavailable columns (mini-MACS columns; Miltenyi Biotec) according to themanufacturer's directions and put into DMEM/Ham's F12/5% FBS withoutnucleosides but with 50 nM methotrexate (selection medium).

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells perwell in 96-well dishes in selection medium and allowed to grow out forapproximately two weeks. The wells are checked for evaporation of mediumand brought back to 200 μl per well as necessary during this process.When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5 mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotatingshaker. The filter is incubated with the antibody-HRP conjugate in 2.5%non-fat dry milk Western A buffer for 1 hour at room temperature on arotating shaker. The filter is then washed three times at roomtemperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filteris developed with chemiluminescence reagents (ECL™ direct labelling kit;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham Corp.) forapproximately 5 minutes. Positive clones are trypsinized from the96-well dish and transferred to 6-well dishes in selection medium forscaleup and analysis by Western blot.

Example 3 Expression in Baby Hamster Kidney Cells

The full-length zcyto24 and zcyto25 proteins were produced in BHK cells.For example, the BHK cells were transfected with eitherzcyto24-CEE/pZMP21 or zcyto25-CEE/pZMP21 (Example 1). BHK 570 cells(ATCC CRL-10314) were plated in T75 tissue culture flasks and allowed togrow to approximately 50 to 70% confluence overnight at 37° C., 5% CO₂,in growth medium (SL7V4, 5% fetal bovine serum (Hyclone, Logan, Utah),1% penicillin/streptomycin). The cells were then transfected withzcyto24-CEE/pZMP21 or zcyto25-CEE/pZMP21 by liposome-mediatedtransfection (using Lipofectamine™; Life Technologies), in serum free(SF) media. The plasmid was diluted into 1.5-ml tubes to a total finalvolume of 640 μl with SF media. Thirty-five μl of the lipid mixture wasmixed with 605 μl of SF medium, and the resulting mixture is allowed toincubate approximately 30 minutes at room temperature. Six ml of SFmedia was then added to the DNA/lipid mixture. The cells were rinsedonce with 5 ml of SF media, aspirated, and the DNA/lipid mixture wasadded. The cells are incubated at 37° C. for five hours, then 15 ml ofgrowth medium was added to each plate. The plates were incubated at 37°C. overnight, and the DNA/lipid mixture was replaced with selectionmedium (SL7V4, 5% fetal bovine serum (Hyclone, Logan, Utah), 1%penicillin/streptomycin, 1 μM MTX) the next day. Approximately 7-10 dayspost-transfection, methotrexate-resistant colonies were trypsinized, andthe cells were re-plated into T-162 flasks and transferred tolarge-scale culture.

Example 4 Construction of Adenovirus Vectors

For construction of adenovirus vectors, the protein coding region ofzcyto20, zcyto21, zcyto22, zcyto24 or zcyto25 is amplified by PCR usingprimers that add PmeI and AscI restriction sites at the 5′ and 3′termini respectively. Amplification is performed with a full-length cDNAtemplate in a PCR reaction as follows: one cycle at 95° C. for 5minutes; followed by 15 cycles at 95° C. for 1 min., 61° C. for 1 min.,and 72° C. for 1.5 min.; followed by 72° C. for 7 min.; followed by a 4°C. soak. The PCR reaction product is loaded onto a 1.2%low-melting-temperature agarose gel in TAE buffer (0.04 M Tris-acetate,0.001 M EDTA). The PCR product is excised from the gel and purifiedusing a commercially available kit comprising a silica gel membrane spincolumn (QIAquick® PCR Purification Kit and gel cleanup kit; Qiagen,Inc.) as per kit instructions. The PCR product is then digested withPmeI and AscI, phenol/chloroform extracted, EtOH precipitated, andrehydrated in 20 ml TE (Tris/EDTA pH 8). The zcyto20 fragment is thenligated into the PmeI-AscI sites of the transgenic vector pTG12-8 andtransformed into E. coli DH10B™ competent cells by electroporation.Vector pTG12-8 was derived from p2999B4 (Palmiter et al., Mol Cell Biol.13:5266-5275, 1993) by insertion of a rat insulin II intron (ca. 200 bp)and polylinker (Fse I/Pme I/Asc I) into the Nru I site. The vectorcomprises a mouse metallothionein (MT-1) promoter (ca. 750 bp) and humangrowth hormone (hGH) untranslated region and polyadenylation signal (ca.650 bp) flanked by 10 kb of MT-1 5′ flanking sequence and 7 kb of MT-13′ flanking sequence. The cDNA is inserted between the insulin II andhGH sequences. Clones containing the zcyto20, zcyto21, zcyto22, zcyto24or zcyto25 cDNA are identified by plasmid DNA miniprep followed bydigestion with PmeI and AscI. Positive clones are sequenced to insurethat there were no deletions or other anomalies in the construct.

DNA is prepared using a commercially available kit (Maxi Kit, Qiagen,Inc.), and the cDNA is released from the pTG12-8 vector using PmeI andAscI enzymes. The cDNA is isolated on a 1% low melting temperatureagarose gel and excised from the gel. The gel slice is melted at 70° C.,and the DNA is extracted twice with an equal volume of Tris-bufferedphenol, precipitated with EtOH, and resuspended in 10 μl H₂O.

The cDNA is cloned into the EcoRV-AscI sites of a modified pAdTrack-CMV(He, T-C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998). Thisconstruct contains the green fluorescent protein (GFP) marker gene. TheCMV promoter driving GFP expression is replaced with the SV40 promoter,and the SV40 polyadenylation signal is replaced with the human growthhormone polyadenylation signal. In addition, the native polylinker isreplaced with FseI, EcoRV, and AscI sites. This modified form ofpAdTrack-CMV is named pZyTrack. Ligation is performed using acommercially available DNA ligation and screening kit (Fast-Link® kit;Epicentre Technologies, Madison, Wis.). Clones containing zcyto20 areidentified by digestion of mini prep DNA with FseI and AscI. In order tolinearize the plasmid, approximately 5 μg of the resulting pZyTrackzcyto20, zcyto21, zcyto22, zcyto24 or zcyto25 plasmid is digested withPmeI. Approximately 1 μg of the linearized plasmid is cotransformed with200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli BJ5183cells (He et al., ibid.). The co-transformation is done using a Bio-RadGene Pulser at 2.5 kV, 200 ohms and 25 pFa. The entire co-transformationmixture is plated on 4 LB plates containing 25 μg/ml kanamycin. Thesmallest colonies are picked and expanded in LB/kanamycin, andrecombinant adenovirus DNA is identified by standard DNA miniprepprocedures. The recombinant adenovirus miniprep DNA is transformed intoE. coli DH10B™ competent cells, and DNA is prepared using a Maxi Kit(Qiagen, Inc.) according to kit instructions.

Approximately 5 μg of recombinant adenoviral DNA is digested with PacIenzyme (New England Biolabs) for 3 hours at 37° C. in a reaction volumeof 100 μl containing 20-30U of PacI. The digested DNA is extracted twicewith an equal volume of phenol/chloroform and precipitated with ethanol.The DNA pellet is resuspended in 10 μl distilled water. A T25 flask ofQBI-293 A cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada),inoculated the day before and grown to 60-70% confluence, is transfectedwith the PacI digested DNA. The PacI-digested DNA is diluted up to atotal volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES). In aseparate tube, 20 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP)(Boehringer Mannheim, Indianapolis, Ind.) is diluted to a total volumeof 100 μl with HBS. The DNA is added to the DOTAP, mixed gently bypipetting up and down, and left at room temperature for 15 minutes. Themedia is removed from the 293 A cells and washed with 5 ml serum-freeminimum essential medium (MEM) alpha containing 1 mM sodium pyruvate,0.1 mM MEM non-essential amino acids, and 25 mM HEPES buffer (reagentsobtained from Life Technologies, Gaithersburg, Md.). Five ml ofserum-free MEM is added to the 293 A cells and held at 37° C. TheDNA/lipid mixture is added drop-wise to the T25 flask of 293 A cells,mixed gently, and incubated at 37° C. for 4 hours. After 4 hours themedia containing the DNA/lipid mixture is aspirated off and replacedwith 5 ml complete MEM containing 5% fetal bovine serum. The transfectedcells are monitored for GFP expression and formation of foci (viralplaques).

Seven days after transfection of 293 A cells with the recombinantadenoviral DNA, the cells express the GFP protein and start to form foci(viral “plaques”). The crude viral lysate is collected using a cellscraper to collect all of the 293 A cells. The lysate is transferred toa 50-ml conical tube. To release most of the virus particles from thecells, three freeze/thaw cycles are done in a dry ice/ethanol bath and a37° C. waterbath.

The crude lysate is amplified (Primary (1°) amplification) to obtain aworking “stock” of zcyto20 rAdV lysate. Ten 10 cm plates of nearlyconfluent (80-90%) 293 A cells are set up 20 hours previously, 200 ml ofcrude rAdV lysate is added to each 10-cm plate, and the cells aremonitored for 48 to 72 hours for CPE (cytopathic effect) under the whitelight microscope and expression of GFP under the fluorescent microscope.When all of the 293 A cells show CPE, this stock lysate is collected andfreeze/thaw cycles performed as described above.

A secondary (2°) amplification of zcyto20 rAdV is then performed. Twenty15-cm tissue culture dishes of 293 A cells are prepared so that thecells are 80-90% confluent. All but 20 ml of 5% MEM media is removed,and each dish is inoculated with 300-500 ml of the 10 amplified rAdvlysate. After 48 hours the 293 A cells are lysed from virus production,the lysate is collected into 250-ml polypropylene centrifuge bottles,and the rAdV is purified.

NP-40 detergent is added to a final concentration of 0.5% to the bottlesof crude lysate in order to lyse all cells. Bottles are placed on arotating platform for 10 minutes agitating as fast as possible withoutthe bottles falling over. The debris is pelleted by centrifugation at20,000× G for 15 minutes. The supernatant is transferred to 250-mlpolycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 MNaCl solution is added. The bottles are shaken overnight on ice. Thebottles are centrifuged at 20,000× G for 15 minutes, and the supernatantis discarded into a bleach solution. Using a sterile cell scraper, thewhite, virus/PEG precipitate from 2 bottles is resuspended in 2.5 mlPBS. The resulting virus solution is placed in 2-ml microcentrifugetubes and centrifuged at 14,000× G in the microcentrifuge for 10 minutesto remove any additional cell debris. The supernatant from the 2-mlmicrocentrifuge tubes is transferred into a 15-ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with CsCl. The solution istransferred to 3.2-ml, polycarbonate, thick-walled centrifuge tubes andspun at 348,000× G for 3-4 hours at 25 μC. The virus forms a white band.Using wide-bore pipette tips, the virus band is collected.

A commercially available ion-exchange column (e.g., PD-10 columnsprepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway, N.J.) isused to desalt the virus preparation. The column is equilibrated with 20ml of PBS. The virus is loaded and allowed to run into the column. 5 mlof PBS is added to the column, and fractions of 8-10 drops arecollected. The optical densities of 1:50 dilutions of each fraction aredetermined at 260 nm on a spectrophotometer. Peak fractions are pooled,and the optical density (OD) of a 1:25 dilution is determined. OD isconverted to virus concentration using the formula: (OD at 260nm)(25)(1.1×10¹²)=virions/ml.

To store the virus, glycerol is added to the purified virus to a finalconcentration of 15%, mixed gently but effectively, and stored inaliquots at −80 μC.

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada)is followed to measure recombinant virus infectivity. Briefly, two96-well tissue culture plates are seeded with 1×10⁴ 293 A cells per wellin MEM containing 2% fetal bovine serum for each recombinant virus to beassayed. After 24 hours 10-fold dilutions of each virus from 1×10⁻² to1×10⁻¹⁴ are made in MEM containing 2% fetal bovine serum. 100 μl of eachdilution is placed in each of 20 wells. After 5 days at 37° C., wellsare read either positive or negative for CPE, and a value for “PlaqueForming Units/ml” (PFU) is calculated.

Example 5 Cloning of Zcyto20, Zcyto22, Zcyto24, and Zcyto25

A: Zcyto20 and Zcyto22

PCR primers were designed that were common to both zcyto20 and zcyto22within the predicted coding sequence. These are designated ZC39339 (SEQID NO:47) and ZC39393 (SEQ ID NO:48). PCR was carried out on a panel ofhuman cDNA libraries from different tissues. The product was observed inbrain, islet (pancreas), prostate, placenta, testis, HPVS (prostateepithelia), and CD3+ libraries. PCR primers were then designed from thegenomic sequence 5′ of the starting methionine and 3′ of the terminationcodon for zcyto20 (with high similarity to zcyto22). These weredesignated ZC39340 (SEQ ID NO:49) and ZC39341 (SEQ ID NO:50). PCR wascarried out on the previously identified libraries. Four librariescontained full length clones. Sequencing of PCR products generated fromthese libraries resulted in three libraries containing zcyto22(prostate, testis and CD3+), and one library containing zcyto20 (HPVS(prostate epithelia)). PCR using primers specific to the predictedcoding sequence of zcyto22 was also carried out. These are designatedZC39295 (SEQ ID NO:51) and ZC39298 (SEQ ID NO:52). Libraries positive bythis PCR were brain, prostate, CD3+, and testis. Sequencing confirmedthe zcyto22 sequence.

B: Zcyto24 and Zcyto25

PCR primers were designed that were common to both zcyto24 and zcyto25within the predicted coding sequence. These are designated ZC39687 (SEQID NO:53) and ZC39741 (SEQ ID NO:54). PCR was carried out on a panel ofmouse cDNA libraries from different tissues. The product was observed inthe following libraries: heart, lung, placenta, Torres prostate, skin,small intestine, testis, and thymus. PCR primers were then designed 5′of the starting methionine and at the termination codons. The 5′ primeris designated ZC39732 (SEQ ID NO:55) and matches both zcyto24 andzcyto25 sequences. The zcyto24 3′ primer is designated ZC39701 (SEQ IDNO:56). The zcyto25 3′ primer is designated ZC39688 (SEQ ID NO:57). PCRwas carried out on the positive libraries indicated above. For zcyto24,all libraries except heart and Torres prostate were positive. Sequencingconfirmed zcyto24 sequence from placenta, testis, and small intestinelibraries. For zcyto25, all libraries except heart and Torres prostatewere positive. Sequencing confirmed zcyto25 sequence from the lunglibrary.

Example 6 Expression in Baculovirus

A: Construct for Expression of Zcyto20

An expression vector, pzBV37L:zCyto20, was prepared to express zcyto20polypeptides in insect cells. A 536 bp fragment containing sequence forzcyto20 and encoded BspE1 and Xba1 restriction sites on the 5′ and 3′ends, respectively, was generated by PCR amplification from a plasmidzcyto20 using primers ZC41932 (SEQ ID NO:14) and ZC41933 (SEQ ID NO:15)utilizing the Expand High Fidelity PCR System (Boerhinger Mannheim) asper manufacturer's instructions. The PCR conditions were as follows: 1cycle of 94° C. for 4 minutes, 30 cycles of 94° C. for 30 seconds, 50°C. for 30 seconds, and 72° C. for 1 minute; 1 cycle at 72° C. for 4 min;followed by a 4° C. soak. A small portion of the PCR product wasvisualized by gel electrophoresis (1% NuSieve agarose), and a fragmentlength of approximately 550 bp was confirmed. The remainder of thereaction mix was purified via the Qiagen PCR purification kit as permanufacturers instructions and eluted in 30 μl water. The cDNA wasdigested in a 36 μl vol. using Bspe1 and Xba1 (New England Biolabs,Beverly, Mass.) in appropriate buffer conditions at 37° C. The digestedPCR product band was run through a 1% agarose TAE gel, excised andextracted using a QIAquick™ Gel Extraction Kit (Qiagen, Cat. No. 28704)and eluted in 30 μL of EB buffer. The digested zcyto20 PCR product wasligated into the multiple cloning site (MCS) of vector pZBV37L at theBspe1 and Xba1 sites. The pZBV37L vector is a modification of thepFastBac1™ (Life Technologies) expression vector, where the polyhedronpromoter has been removed and replaced with the late activating BasicProtein Promoter and the EGT leader signal sequence upstream of the MCS.5 μL of the restriction enzyme digested zcyto20 PCR fragment and 4 μL ofthe corresponding pZBV37L vector were ligated for 72 hours at 15° C. ina 20 μL vol. in appropriate buffer conditions. 5 μL of the ligation mixwas transformed into 33 μl of ElectoMAX™ DH12S™ cells (LifeTechnologies, Cat. No. 18312-017) by electroporation at 400 Ohms, 2.00kV and 25 pF in a 2 mm gap electroporation cuvette (BTX, Model No. 620).The transformed cells were diluted in 500 μl of LB media and outgrownfor 1 hr at 37° C. and 10 μL and 20 μL of the dilution were plated ontoLB plates containing 100 μg/ml ampicillin. Clones were analyzed by PCRand positive clones were selected, plated, and submitted for sequencing.The sequence was then confirmed.

B. Construction and Expression of Tagged Zcyto20

An expression vector, pZBV32L:zCyto20cee, was prepared to expresszcyto20cee polypeptides in insect cells. PZBV32L:zCyto20cee was designedto express a zcyto20 polypeptide with a C-terminal GLU-GLU tag (SEQ IDNO:16). This construct can be used to determine the N-terminal aminoacid sequence of zcyto20 after the signal peptide has been cleaved off.

1. Construction of pZBV32L:zCyto20cee

A 625 bp zcyto20 fragment containing BamHI and XbaI restriction sites onthe 5′ and 3′ ends, respectively, was generated by PCR amplificationfrom a plasmid containing zcyto20 cDNA using primers zc40240 and zc40241(SEQ ID NOS: 17 and 18, respectively). The PCR reaction conditions wereas follows: The Expand High Fidelity PCR System (Boehringer Mannheim)was utilized for a 100 μl volume reaction containing 5% DMSO. 1 cycle at94° C. for 4 minutes; 30 cycles of 94° C. for 30 seconds, 50° C. for 30seconds, and 72° C. for 60 seconds; 1 cycle at 72° C. for 4 min;followed by 4° C. soak. Five μl of the PCR fragment was visualized bygel electrophoresis (1% NuSieve agarose). The remainder of the reactionmix was purified via Qiagen PCR purification kit as per manufacturersinstructions and eluted in 30 μl water. The cDNA was digested in a 35 μlvolume using BamHI and XbaI (New England Biolabs, Beverly, Mass.) inappropriate buffer conditions for 2 hrs at 37° C. The digested PCRproduct band was run through a 1% agarose TAE gel, excised and extractedusing a QIAquick™ Gel Extraction Kit (Qiagen) and eluted in 30 μl ofwater. The purified, digested zCyto20cee PCR product was ligated intothe multiple cloning site of a previously prepared and restrictionenzyme digested (BamHI and XbaI) vector pZBV32L. The pZBV32L vector is amodification of the pFastBac1™ (Life Technologies) expression vector,where the polyhedron promoter has been removed and replaced with thelate activating Basic Protein Promoter, and the coding sequence for theGlu-Glu tag (SEQ ID NO:10) as well as a stop signal was inserted at the3′ end of the multiple cloning region. Five μl of the restrictiondigested zCyto20 insert and 40 ng of the corresponding pZBV32L vectorwere ligated overnight at 16° C. in a 20 μl vol. Five μl of the ligationmix was transformed into 50 μl of ElectoMAX™ DH12s™ cells (LifeTechnologies) by electroporation at 400 Ohms, 2.00 kV and 25 pF in a 2mm gap electroporation cuvette. The transformed cells were diluted in500 μl of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml1M NaCl, 1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) and 50μl of the dilution were plated onto LB plates containing 100 μg/mlampicillin. Clones were analyzed by PCR and restriction digestion.Positive clones were selected and plated for sequencing. Once propersequence was confirmed, 25 ng of positive clone DNA was transformed into66 μl DH10Bac™ Max Efficiency® competent cells (GIBCO-BRL) by heat shockfor 45 seconds in a 42° C. heat block. The transformed DH10Bac™ cellswere diluted in 600 μl SOC media (2% Bacto Tryptone, 0.5% Bacto YeastExtract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mMglucose) and grown at 37° C. for 1 hr and 100 μl were plated onto LuriaAgar plates containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/mltetracycline, 40 μg/mL IPTG and 200 μg/mL Bluo Gal. The plates wereincubated for 48 hours at 37° C. A color selection was used to identifythose cells having transposed viral DNA (referred to as a “bacmid”).Those colonies, which were white in color, were picked for analysis.Colonies were analyzed by PCR and positive colonies (containing desiredbacmid) were selected for growth and subsequent bacmid DNA purification.Clones were screened for the correct molecular weight insert by PCRamplification of DNA using primers to the transposable element in thebacmid: ZC447 (SEQ ID NO:19) and ZC976 (SEQ ID NO:20). The PCR reactionconditions were as follows: 1 cycle at 94° C. for 4 minutes; 25 cyclesof 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 2.5 min;1 cycle at 72° C. for 4 min; followed by 4° C. soak. The PCR product wasrun on a 1% agarose gel to confirm the insert size. Those having thecorrect size insert were used to transfect Spodoptera Frugiperda (Sf9)cells.

2. Transfection

Sf9 cells were seeded at 1×10⁶ cells per well in a 6-well plate andallowed to attach for 1 hour at 27° C. Approximately 5 μg of bacmid DNAwere diluted to 100 μl with Sf-900 II SFM (Life Technologies). Twenty μlof Lipofectamine™ Reagent (Life Technologies) were diluted to 100 μlwith Sf-900 II SFM. The bacmid DNA and lipid solutions were gently mixedand incubated for 45 minutes at room temperature. Eight hundred μl ofSf-900 II SFM was added to the lipid-DNA mixture. The media wasaspirated from the well and the 1 ml of DNA-lipid mix added to thecells. The cells were incubated at 27° C. overnight. The DNA-lipid mixwas aspirated from each well and was replaced with 2 ml of Sf-900 IImedia. The plates were incubated at 27° C., 90% humidity, forapproximately 7 days after which the virus was harvested.

3. Amplification

Sf9 cells were seeded at 1×10⁶ cells per well in a 6-well plate. Fivehundred μl of virus from the transfection plate were placed in the welland the plate was incubated at 27° C., 90% humidity, for 96 hours afterwhich the virus was harvested (primary amplification).

The second round of amplification was carried out by transferring 100 μlof virus from the above primary amplification plate to wells containing1×10⁶ cells per well. The plate was incubated for 96 hours beforeharvesting.

An additional round of amplification was performed (tertiary amp.) Sf9cells were grown in 50 ml Sf-900 II SFM in a 250 ml shake flask to anapproximate density of 1×10⁶ cells/ml. They were then infected with 1 mlof the viral stock from the above plate and incubated at 27° C. for 6days after which time the virus was harvested.

The viral stock was titered by a growth inhibition curve and the titerculture that indicated a Multiplicity Of Infection (MOI) of one wasallowed to proceed for a total of 48 hrs. The supernatant was analyzedvia a reduced Western using a primary monoclonal antibody specific forthe Glu-Glu tag followed by an HRP-conjugated goat anti-murine secondaryantibody. Results indicated a band of approximately 20 kDa. Supernatantwas also provided for activity analysis.

A large viral stock was then generated by the following method: Sf9cells were grown in IL Sf-900 II SFM in a 2800 ml shake flask to anapproximate density of 1×10⁶ cells/ml. They were then infected with 5 mlof the viral stock from the above flask and incubated at 27° C. for 4days after which time the virus was harvested.

Larger scale infections were completed to provide material fordownstream purification.

Similarly, zcyto21 and zcyto22 were expressed in Baculovirus

Example 7 Purification of Protein

Recombinant protein can be made for any of the protein described herein.

A. Purification of Zcyto20

Recombinant carboxyl terminal Glu-Glu tagged zcyto20 was produced fromeither recombinant baculovirus infected insect cells, stable ortransient BHK cell lines. Cultures were harvested, and the media weresterile filtered using a 0.2 □m filter.

Proteins were purified from the conditioned media by a combination ofAnti-Glu-Glu (Anti-EE) peptide antibody affinity chromatography andSuperdex 75 gel exclusion chromatography. Culture medium from BV (pH6.0, conductivity 7 mS) was adjusted to pH 6.7. Both BV and BHK mediawere then added NaCl to 300 mM before loading onto a 10×70 mm (5-mlcolumn volume) Poros Protein A anti-EE antibody affinity column at aflow of 2-5 ml/minute. The column was then washed with five columnvolumes (CV) of 5×PBS (pH 7.2). Bound protein was eluted with 0.5 Macetic acid, 0.5 M NaCl, pH 3.0. Two-ml fractions were collected, andthe eluant was neutralized by the addition of 2 M Tris. Samples from theanti-EE antibody affinity column were analyzed by SDS-PAGE with silverstaining and western blotting for the presence of zcyto20 protein.Zcyto20 protein-containing fractions were pooled and concentrated toabout 2 mls using Biomax-5 concentrator (Millipore), and loaded onto a16×600 mm Superdex 75 gel filtration column (Amersham PharmaciaBiotech). The fractions containing purified zcyto20 protein were pooled,filtered through 0.2 μm filter, aliquoted into 100 μl each, and frozenat −80° C. The concentration of the final purified protein wasdetermined by BCA assay (Pierce, Rockford, Ill.) and HPLC-amino acidanalysis.

B. SDS-PAGE and Western Blotting Analysis of Zcyto20 Proteins

Recombinant zcyto20 protein was analyzed by SDS-PAGE (Nupage 4-12%Bis-Tris, Invitrogen, Calsbad, Calif.) with silver staining method (FastSilver, Geno Technology, Inc., St. Louis, Mo.) and Western blottingusing the anti-EE antibody. Either the conditioned media or purifiedprotein was electrophoresed using an Xcell II™ MINI-CELL (Invitrogen,Calsbad, Calif.) and transferred to nitrocellulose (0.2 □m; Bio-RadLaboratories, Hercules, Calif.) at room temperature using Xcell II™ blotmodule (Invitrogen) with stirring according to directions provided inthe instrument manual. The transfer was run at 500 mA for 45 minutes ina buffer containing 25 mM Tris base, 200 mM glycine, and 20% methanol.The filters were then blocked with 10% non-fat dry milk in PBS for 10minutes at room temperature. The nitrocellulose was quickly rinsed, andthen primary antibody was added in PBS containing 2.5% non-fat dry milk.The blots were incubated for two hours at room temperature or overnightat 4° C. with gentle shaking. Following the incubation, blots werewashed three times for 10 minutes each in PBS. Secondary antibody(rabbit anti-mouse IgG conjugated to horseradish peroxidase; obtainedfrom Pierce Chemical Co., Rockford, Ill.) diluted 1:2000 in PBScontaining 2.5% non-fat dry milk was added, and the blots were incubatedfor two hours at room temperature with gentle shaking. The blots werethen washed three times, 10 minutes each, in PBS, then quickly rinsed inH₂O. The blots were developed using commercially availablechemiluminescent substrate reagents (SuperSignal® ULTRA reagents 1 and 2mixed 1:1; reagents obtained from Pierce Chemical Co.), and the signalwas captured using Lumi-Imager's Lumi Analyst 3.0 software (BoehringerMannheim GmbH, Germany) for times ranging from 10 second to 5 minutes oras necessary.

C. Summary of Protein Purification and Analysis

The purified zcyto20-CEE protein from BV media migrated as a 21 kDamonomer on the 4-12% Bis-Tris gel, however, a minor 36 kDa dimer bandwas also observed. The dimer protein became monomer band upon reductionusing reducing agent, suggesting the dimerization of zcyto20-CEE bydisulfide bond, and it is consistent with the odd number (a total ofseven) of cysteine residues resulting in interchain disulfide bond.

Zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides were purified in asimilar manner.

Example 8 Interferon-Like Transcriptional Regulatory Regions of Zcyto20

Local sequence alignments of the characterized IFN-α, IFN-β, IFN-γpromoters and those of a number of other cytokines with the upstreamregions of zcyto20, zcyto21, and zcyto24 were performed to identifywhether these genes were co-regulated. The hypothesis was that commonproperties of gene regulation should be reflected in sequencesimilarities of the regions upstream of the genes.

Due to the low binding specificity of TFs, predictions of individualbinding sites have a high rate of false positives. Therefore, the sitepredictions in isolation for the identification of binding sites withfunctional roles in vivo are not useful. However, predicted bindingsites likely to have sequence-specific functions can be selected bymeans of a conservation-based filter: The biological observation thatregulatory regions are often more strongly conserved between speciesthan other non-coding regions can be quantified to reveal patterns ofconservation which have been called phylogenetic footprints (Fickett, etal., Curr. Opin. Biotechnol. 11: 19-24, 2000.) In particular,human-rodent comparisons have proven a valuable resource for theidentification of functional regulatory elements (Wasserman et al., Nat.Genet. 26: 225-228, 2000).

The alignments revealed significant matches mainly among the promotersof the various IFN-αgenes, whereas, based on this analysis, there islittle evidence for similarity between the promoters of zcyto20-22 andother cytokines.

The pairwise sequence alignments were performed with DBA (Jareborg etal., Genome. Res. 9: 815-824, 1999). The search for individualtranscription factor binding sites was performed with standard positionweight matrices (Fickett, Mol. Cell. Biol. 16:437-441, 1996) drawn fromthe TRANSFAC database (version 3.0, Wingender et al., Nucleic Acids Res.28: 316-319, 2000), and alignments of regions 1-4 were computed withSSEARCH, version 3.1t12 (Pearson, Genomics 11:635-650, 1991). Based onpublished studies involving other sets of TFs, most natural bindingsites sufficiently conserved between mouse and human can be expected tobe detected in the used score range (Fickett, Mol. Cell. Biol. 16:437-4411996; Wasserman et al., J. Mol. Biol. 278: 167-181, 1998).

The computational results suggest that features common to the regulationof the zcyto20 family and other cytokines that reflected by sequencesimilarity would be expected on the level of individual binding sitesrather than matches covering extended regions.

Based on a search of binding sites of a set of 32 TFs, theidentification of putative sites of a limited number of factors largelyknown to be involved in the transcriptional regulation of interferonswere made. The results of the comparison show putative binding sites ofTFs playing important roles in the transcriptional regulation of IFN-β[NF-κB, ISRE (element binding IRFs)] and IFN-γ (AP-1, CREB, GATA, NF-κB,NF-AT) are also present in the conserved non-coding regions. Forexample, a pair of neighboring AP-1/NF-AT sites in region 1, is awell-documented example of cooperative binding occurring in manycytokine promoters (Holloway et al., Mol. Immunol. 38: 567-580, 2000).

The binding sites for TFs that have been identified as critical forcytokine regulation, suggest that the cluster of transcription factorbinding sites in the promoter region of zcyto20 is a candidate for afunctional region.

The alignment of region 2 with the group of known cytokines yielded amatch in the AK155 promoter, at position −415 with respect to thetranscription start site of AK155 as given in Knappe et al. (J. Virol.74: 3881-3887, 2000). The fact that there is a putative NF-κB bindingsite at that location in the zcyto20 promoter indicates the possiblepresence of a NF-κB site in the AK155 promoter.

Example 9 Transgenic Animals

Transgenic animals expressing Zcyto21 genes were produced using adult,fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms,Germantown, N.Y.)), vasectomized males (duds) (CD1, 2-8 months, (TaconicFarms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks,(Taconic Farms)) and adult fertile females (recipients) (CD1, 2-4months, (Taconic Farms)).

The donors were acclimated for 1 week and then injected withapproximately 8 IU/mouse of Pregnant Mwere's Serum gonadotrophin (Sigma,St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors were mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation wasconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs were collected under a surgical scope (Leica MZ12 StereoMicroscope, Leica, Wetzlar, Del.). The oviducts were collected and eggswere released into urinanalysis slides containing hyaluronidase (Sigma).Eggs were washed once in hyaluronidase, and twice in Whitten's W640medium (Table 7) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂at 37° C. The eggs were then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a cDNA of the Zcyto21gene was linearized, gel-purified, and resuspended in 10 mM Tris pH 7.4,0.25 mM EDTA pH 8.0, at a final concentration of 5-10 nanograms permicroliter for microinjection.

Plasmid DNA was microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA wasdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg was penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA were injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure was repeated until all the eggs were injected. Successfullymicroinjected eggs were transferred into an organ tissue-culture dishwith pregassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, 12-17 healthy 2-cell embryos from the previous day'sinjection were transferred into the recipient. The swollen ampulla waslocated and holding the oviduct between the ampulla and the bursa, anick in the oviduct was made with a 28 g needle close to the bursa,making sure not to tear the ampulla or the bursa. The embryos wereimplanted through this nick, and by holding onto the peritoneal wall,the reproductive organs were guided back into the abdominal cavity.

The recipients were returned to cages in pairs, and allowed 19-21 daysgestation. Animals injected with the Zcyto21 cDNA died before birth.

TABLE 7 WHITTEN'S 640 MEDIA mgs/200 m mgs/500/ml NaCl 1280 3200 KCl 72180 KH₂PO₄ 32 80 MgSO₄•7H₂O 60 150 Glucose 200 500 Ca²⁺ Lactate 106 265K Penn 15 37.5 Streptomycin SO₄ 10 25 NaHCO₃ 380 950 Na Pyruvate 5 12.5H₂0 200 500 EDTA 100 μl 250 μl 5% Phenol Red 200 μl 500 μl BSA 600 1500All reagents are available from Sigma.

Example 10 Antibody Production

A: Zcytor19 Polyclonal Antibodies

Polyclonal antibodies are prepared by immunizing 2 female New Zealandwhite rabbits with the purified recombinant protein huzcytor19/MBP-6H.The rabbits are each given an initial intraperitoneal (ip) injection of200 μg of purified protein in Complete Freund's Adjuvant followed bybooster ip injections of 100 μg peptide in Incomplete Freund's Adjuvantevery three weeks. Seven to ten days after the administration of thesecond booster injection (3 total injections), the animals are bled andthe serum is collected. The animals are then boosted and bled everythree weeks.

The huzcyotr19/MBP-6H specific rabbit serum is pre-adsorbed of anti-MBPantibodies using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB,Peapack, N.J.) that is prepared using 10 mg of purified recombinant MBPper gram of CNBr-SEPHAROSE. The huzcytor19-specific polyclonalantibodies are affinity purified from the rabbit serum using aCNBr-SEPHAROSE 4B protein column that is prepared using 10 mg of thespecific antigen purified recombinant protein huzcytor19/MBP-6H followedby 20× dialysis in PBS overnight. Huzcytor19-specific antibodies arecharacterized by ELISA using 500 ng/ml of the purified recombinantproteins huzcytor19/MBP-6H or huzcytor19-Fc4 as antibody targets. Thelower limit of detection (LLD) of the rabbit anti-huzcytor19/MBP-6Haffinity purified antibody on its specific purified recombinant antigenhuzcytor19/MBP-6H and on purified recombinant huzcytor19-Fc4 isdetermined.

B: Zcyto20 Zcyto21 Zcyto22 Zcyto24 and Zcyto25 Polyclonal Antibodies

Polyclonal antibodies are prepared by immunizing female New Zealandwhite rabbits with the purified recombinant protein zcyto20/MBP-6H,zcyto21/MBP-6H, and zcyto22/MBP-6H, as well as mouse zcyto24/MBP-6H, orzcyto25/MBP-6H. The rabbits are each given an initial intraperitoneal(ip) injection of 200 μg of purified protein in Complete Freund'sAdjuvant followed by booster ip injections of 100 μg peptide inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the second booster injection (3 total injections),the animals are bled and the serum is collected. The animals are thenboosted and bled every three weeks.

The zcyto20/MBP-6H, zcyto21/MBP-6H, zcyto22/MBP-6H, zcyto24/MBP-6H, orzcyto25/MBP-6H specific rabbit serum can be pre-adsorbed of anti-MBPantibodies using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB,Peapack, N.J.) that is prepared using 10 mg of purified recombinant MBPper gram of CNBr-SEPHAROSE. The zcyto20, zcyto21, zcyto22, zcyto24, orzcyto25-specific polyclonal antibodies are affinity purified from therabbit serum using a CNBr-SEPHAROSE 4B protein column that is preparedusing 10 mg of the specific antigen purified recombinant proteinfollowed by 20× dialysis in PBS overnight. Antibodies are characterizedby ELISA using 500 ng/ml of the purified recombinant proteins asantibody targets. The lower limit of detection (LLD) of the purifiedantibody on its specific purified recombinant antigen is determined.

Polyclonal antibodies were prepared for zcyto21 using a similar protocolby immunizing rabbits with zcyto21-CEE tagged protein, and theantibodies were purified.

Example 11 Expression of the Zcyto20 Zcyto211 Zcyto22 Zcyto24 andZcyto25 Genes Following Polyinosinic Acid-Polycytidylic Acid Induction

A: Various Cell Types

Cultures of primary cells (normal human bronchial epithelial cells,normal human dermal fibroblasts, human umbilical vein endothelial cells,human microvascular endothelial cells and human smooth muscle cells;CLONETICS Corporation; Walkersville, Md.) and human choriocarcinoma celllines (Jar, BeWo, and 3A-Sub E cells; ATCC, Manassas, Va.) were grown inthe presence of polyinosinic acid-polycytidylic acid (poly I:C; 100ug/ml) (SIGMA; St. Louis, Mo.) or in medium alone. In some instances 10ng/ml hTNFa or 10 ng/ml hIL1b were also tested. After four hours ofincubation, total RNA was isolated from cells and treated withRNase-free DNase. One microgram of total RNA was used for first-standcDNA synthesis using the Advantage RT-for-PCR kit as suggested by themanufacturer (Clontech, Palo Alto, Calif.). Five percent of the cDNAreaction was used for polymerase chain reaction as suggested by themanufacturer (Clontech) using the following primer pairs: zcyto20,ZC40134 (SEQ ID NO:30), ZC40214 (SEQ ID NO:31); zcyto21, ZC40209 (SEQ IDNO:32), ZC40213 (SEQ ID NO:33); zcyto22, ZC39295 (SEQ ID NO:34), andZC39298 (SEQ ID NO:35). Primers to G3PDH were used as a control.

Zcyto20mRNA was detected at low levels in all cell types tested.Increased zcyto20mRNA levels were seen in in NHBE, HUVEC, JAR, 3-A Sub Eand BeWo cells stimulated with poly I:C. Zcyto22 mRNA was detected atlow levels in all cell types tested. Increased zcyto22 mRNA levels wereseen in in NHBE, JAR, 3-A Sub E and BeWo cells stimulated with poly I:C.These results indicate that zcyto20 and zcyto22 mRNA synthesis isstimulated by the known interferon inducer, poly I:C.

Zcyto21 mRNA was detected at low levels in all cell types tested.Increased zcyto21 mRNA levels were seen in in NHBE, HUVEC, NHDF, SMC,HMVEC, JAR, 3-A Sub E and BeWo cells stimulated with poly I:C. Increasedzcyto21 mRNA levels were also seen in IL1b-treated 3-A Sub E placentalcells. These results indicate that zcyto21 mRNA synthesis is stimulatedby the known interferon inducer, poly I:C and can also be stimulated bythe cytokine IL1b in certain cell types.

B: Peripheral blood mononuclear cells

Whole peripheral blood mononuclear cells were isolated from human bloodusing Ficoll Hypaque. T-cells were were purified from peripheral bloodmononuclear cells by VarioMacs positive selection columns as permanufacturer's instructions (Miltenyi Biotec Inc., Auburn, Calif.).Samples from each population were stained and analyzed by fluorescentantibody cell sorting (FACS) (Bectin Dickinson, San Jose, Calif.)analysis to determine the percent enrichment. The CD3+ T-cells wereapproximately 95% purified. Whole peripheral blood leukocytes or CD3+ Tcells were grown in the presence of poly I:C (100 ug/ml) or in mediumalone. After four hours of incubation, total RNA was isolated from cellsand treated with RNAse-free DNAse. RT-PCR was performed with theSuperscript One-Step RT-PCR with Platinum Taq kit (Invitrogen,Frederick, Md.) using 100 ng total RNA as a template for cDNA synthesis.The PCR primer pairs used were: zcyto20: ZC40134 (SEQ ID NO:30) andZC40214 (SEQ ID NO:31); zcyto21: ZC40209 (SEQ ID NO:32) and ZC40213 (SEQID NO:33); zcyto22: ZC39295 (SEQ ID NO:34) and ZC39298 (SEQ ID NO:35).Aliquots of each RNA, were also tested with primer pairs specific forMHC Class I (Clontech) as a control.

Zcyto20 mRNA was detected in whole peripheral blood mononuclear cellsstimulated with poly I:C. The results indicate that zcyto20 mRNAsynthesis is stimulated by the known interferon inducer, poly I:C inperipheral blood mononuclear cells.

Zcyto21 mRNA was detected in whole peripheral blood mononuclear cellsand CD3+ T-cells stimulated with poly I:C. The results indicate thatzcyto21 mRNA synthesis is stimulated by the known interferon inducer,poly I:C in peripheral blood mononuclear cells including CD3+ T cells.

Zcyto22 mRNA was detected in whole peripheral blood mononuclear cellsand CD3+ T-cells stimulated with poly I:C. The results indicate thatzcyto22 mRNA synthesis is stimulated by the known interferon inducer,poly I:C in peripheral blood mononuclear cells including CD3+ T cells.

These results are indicative of the effect poly I:C would have onzcyto24 and zcyto25 as well as other family members.

Example 12 Expression Analysis of Human Primary Immune Cell and ImmuneCell Lines Using RT-PCR

A panel of RNAs from primary human immune cell populations and humanimmune cell lines was screened for zcyto21, zcyto21, and zcyto22expression using RT-PCR. The panels were made in house and contained RNAfrom sixteen various resting and activated cells as described below. Allprimary immune cell populations were isolated from the blood of severalanonymous donors. Various immune cell subsets (CD3+, CD14+, CD19+, andCD56+) were then isolated using labeled Microbeads and the Magnetic CellSeparation System from Miltenyi Biotec. RNA was prepared using theRNeasy Midiprep™ Kit (Qiagen, Valencia, Calif.) as per manufacturer'sinstruction. CD56+ NK cell RNA was isolated from cells in their restingstate. One CD3+ population was activated using a combination of 500ng/ml Ionomycin and 5.0 ng/ml PMA (phorbol 12-myristate 13 acetate).Another CD3+ population was stimulated using the supernatant fromConconavalin A stimulated rat splenocytes, a media known to be rich incytokines and growth factors. CD3+ cells were collected for RNAisolation at activation times of 0, 4 and 16 hours. The CD 19+ sampleswere isolated from human tonsil and activated with 0.5 ug/ml ionomycinand 10 ng/ml PMA. Cells were then collected at 0, 4 hours and 24 hoursand RNA isolated. Human CD14+ monocytes were activated with either 0.1ng/ml lipopolysaccharide (LPS) or 1.0 ng/ml LPS for 20 hours. Restingand activated cells were then collected and the RNA was isolated. Inaddition, RNA was isolated from resting and activated human monocytecell lines HL-60, THP-1 and U937. HL-60 cells were activated overnightwith 10 ng/ml PMA. THP-1 cells were activated overnight with 1.0 ng/mlLPS+10 ng/ml IFNgamma. Finally, U937 cells were activated overnight with10 ng/ml PMA. RT-PCR was performed with the Superscript One-Step RT-PCRwith Platinum Taq kit (Invitrogen) using 100 ng total RNA as a templatefor cDNA synthesis. The PCR primer pairs used were: zcyto20: ZC40632(SEQ ID NO:36) and ZC40633 (SEQ ID NO:37); zcyto21: ZC40209 (SEQ IDNO:32) and ZC40213 (SEQ ID NO:33); and zcyto22: ZC40638 (SEQ ID NO:38)and ZC40639 (SEQ ID NO:39). Aliquots of each RNA, were also tested withprimer pairs specific for MHC Class I (Clontech) as a control.

Zcyto20 mRNA was detected in THP-1 cells treated with LPS and interferongamma. Zcyto20 mRNA was also detected in CD3+ cells treated with PMA for4 h. Zcyto21 mRNA was detected at low levels in resting U937 cells andresting THP-1 cells. The level of zcyto21 mRNA was increased upontreatment of the THP-1 cells with LPS and interferon gamma. Zcyto21 mRNAwas also detected in CD3+ cells treated with PMA for 4 h. Zcyto22 mRNAwas detected in THP-1 cells treated with LPS and interferon gamma.Zcyto22 mRNA was also detected in CD3+ cells treated with PMA for 4 hand 16 h as well as CD3+ cells treated with Conconavalin A for 4 and 16h. The results indicate that zcyto20, zcyto21, and zcyto22 mRNAsynthesis is stimulated by activation of a monocyte cell line andprimary CD3+ immune cells.

These results are indicative of the effect poly I:C would have onzcyto24 and zcyto25 as well as other family members.

Example 13 Antiviral Activity: Cytopathic Effect in Hela and L929 Cells

Initial functional assays for antiviral activity were conducted usingconditioned media from transiently transfected human embryonal kidney(HEK) cells. Production of this conditioned medium is described asfollows. A full-length cDNA for zcyto20, zcyto21, zcyto22, zcyto24, orzcyto25 was cloned into the pzp7Z vector using standard procedures. Thezcyto20, zcyto21, zcyto22, zcyto24, or zcyto25 constructs weretransfected into 293 HEK cells. Briefly, for each construct 700,000cells/well (6 well plates) were plated approximately 18 h prior totransfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1.5micrograms zcyto20, zcyto21, zcyto22, zcyto24, or zcyto25 DNA and 0.5micrograms pIRES2-EGFP DNA (Clontech) were added to 6 microliters Fugene6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. Twomicrograms pIRES2-EGFP DNA alone was used as a negative control. Thesetransfection mixtures were added 30 minutes later to the pre-plated 293cells. Twenty-four hours later the cell media were removed and DMEM+0.1%bovine serum albumin was added. Conditioned media was collected after 48hours, filtered through a 0.45 micron filter and used for antiviral andreporter assays.

Antiviral Assays were carried out using human cervical carcinoma cells(HeLa) and mouse fibroblast cells (L929). On the first day, conditionedmedium containing zcyto20, zcyto21, zcyto22, zcyto24, or zcyto25 (SeeExample 10) was diluted and plated with 50,000 cells in a 96-well flatbottom microtiter plate. Following a 24-hour incubation at 37° C., themedium was removed and replaced with medium containingencephelomyocarditis virus at a multiplicity of infection of 0.1. Thecells were again incubated for 24 hours at 37° C. Culture wells werethen scored visually on a 4-point scale for the presence of cytopathiceffect, which was then converted to % CPE as shown in Table 8.Conditioned medium from cells transfected with GFP alone and purifiedhuman interferon-a-2a or murine interferon-alpha were included ascontrols.

TABLE 8 Determination of Cytopathic Effect Designation Observation ofCytopathic Effect (CPE) — No CPE +/− Possible CPE (about 1% of monolayersurface) + CPE limited to one plaque (about 5% of the surface) +1   CPEis limited to three plaques, affecting less than 25% of the monolayer 125% CPE 1-2 37% CPE 2 50% CPE 2-3 62% CPE 3 75% CPE 3-4 87% CPE 4 100%CPE

Table 9 shows that conditioned medium containing zcyto20, zcyto21,zcyto22, zcyto24, and zcyto25 inhibited viral infection (% CPE) in HeLacells in a dose-dependent manner, while control GFP conditioned mediumfailed to significantly block the appearance of cytopathic effect. Asshown in Table 10, conditioned medium containing zcyto20, zcyto21,zcyto22, zcyto24, and zcyto25 did not inhibit viral infection in L929cells. In both experiments purified interferon showed positive antiviralactivity.

TABLE 9 Percentage Cytopathic Effect of zcyto20, zcyto21, zcyto22,zcyto24, and zcyto25 in HeLa Cells using Conditioned Medium (CM)Relative CM Control zcyto20 zcyto21 zcyto22 zcyto24 zcyto25 hIFN-hIFN-a-2a Concentration GFP (CM) (CM) (CM) (CM) (CM) a-2a ConcentrationNo Add 87 87 87 87 87 87 87 0 ng/ml .008X 87 10 56 0 0 10 15 .0001 ng/ml.0156X 87 2.5 31 0 0 5 8.3 .001 ng/ml .0325X 87 5 10 0 0 5 1.7 .01 ng/ml.0625X 87 2.5 10 0 0 0 0 .1 ng/ml .125X 87 0 5 0 0 0 0 1 ng/ml .25X 87 00 0 0 0 0 10 ng/ml .5X 87 0 0 0 0 0 0 100 ng/ml

TABLE 10 Percentage Cytopathic Effect of zcyto20, zcyto21, zcyto22,zcyto24, and zcyto25 in L929 Cells using Conditioned Medium (CM)Relative CM Control zcyto20 zcyto21 zcyto22 zcyto24 zcyto25 mIFN-mIFN-alpha Conc. GFP (CM) (CM) (CM) (CM) (CM) alpha Conc. No Add 87 8787 87 87 87 87 0 ng/ml .008X 87 87 87 87 87 87 87 .0001 ng/ml .0156X 8787 87 87 87 87 87 .001 ng/ml .0325X 87 87 87 87 87 87 87 .01 ng/ml.0625X 87 87 87 87 87 87 58 .1 ng/ml .125X 87 87 87 87 87 87 6.7 1 ng/ml.25X 87 87 87 87 87 87 0 10 ng/ml .5X 87 87 87 87 87 87 0 100 ng/ml

As a follow up, conditioned medium from Sf9 cells infected withbaculovirus expressing zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25was used in antiviral assays. Conditioned medium from Sf9 cells infectedwith wild type baculovirus was used as a negative control.

The results of the antiviral assay using the baculovirus-derivedconditioned medium were similar to that using the 293 transienttransfected conditioned medium. Table 11 shows that baculovirus-derivedconditioned medium containing zcyto21 inhibited viral infection in HeLacells in a dose-dependent manner, while control baculovirus conditionedmedium failed to block the appearance of cytopathic effect.

TABLE 11 Percentage Cytopathic Effect in HeLa Cells usingBaculovirus-derived zcyto21 Conditioned Medium (CM) Relative CM zcyto21Control hIFN-a-2a Concentration CM BV CM hIFN-a-2a Concentration No Add51 51 51 0 ng/ml .008X 5.5 56 17.5 .001 ng/ml .0156X 2.5 62 7.5 .01ng/ml .0325X 5 56 0 .1 ng/ml .0625X 2.5 50 0 1 ng/ml .125X 2.5 56 0 10ng/ml .25X 5 62 0 100 ng/ml

The production of the baculovirus construct and conditioned medium isdescribed above.

Example 14 Antiviral Activity can not be Blocked by Antibodies Againstthe Human Interferon Alpha Receptor Chain 2 Beta

Additional antiviral assays were carried out using human cervicalcarcinoma cells (HeLa). On the first day, anti-hu-IFN-Receptor MAb(Research Diagnostics Inc) and Isotype-matched negative control MAb werediluted into a 96-well flat bottom microtiter plate. Conditioned mediumfrom Sf9 cells infected with baculovirus expressing zcyto20, zcyto21, orzcyto22 was added to obtain a final concentration of 0.0625×CM andplated with 50,000 HeLa cells per well. Following a 24-hour incubationat 37° C., the medium was removed and replaced with medium containingencephelomyocarditis virus at a multiplicity of infection of 0.1. Thecells were again incubated for 24 hours at 37° C. Culture wells werethen scored visually for the presence of cytopathic effect (CPE), asshown in Table 8. Purified human interferon-a-2a at a concentration of0.01 ng/ml was included as a positive control.

Table 12 shows that conditioned medium containing zcyto20, zcyto21, andzcyto22 had antiviral activity in HeLa cells (as indicated by zero %CPE) in the presence or absence of neutralizing antibodies against thehuman interferon alpha receptor chain 2 beta. In contrast, the antiviralactivity of interferon-a-2a was inhibited in a dose-dependent mannerspecifically in the presence of neutralizing antibodies against thehuman interferon alpha receptor chain 2 beta. These data indicate thatzcyto20 zcyto21, and zcyto22 interact with either a receptor other thanthe human interferon alpha receptor or it interacts with the humaninterferon alpha receptor with a different mechanism than humaninterferon-a-2a.

TABLE 12 Percentage Cytopathic Effect in HeLa Cells using zcyto20,zcyto21, or zcyto22 Conditioned Medium (CM) and Neutralizing Antibodiesagainst the Human Interferon Alpha Receptor Chain 2 Beta zcyto20 Anti-CM zcyto21 + CM zcyto22 + CM zcyto22 + hIFN-a- + hIFNR Anti- zcyto20 +CM Anti- zcyto21 + CM Anti- CM 2a + Anti- hIFN-a- MAb hIFNR ControlhIFNR Control hIFNR Control hIFNR 2a + Control Concentration MAb MAb MAbMAb MAb MAb Mab MAb 0 ug/ml 0 0 0 0 0 0 7.5 10 0.001 ug/ml 0 0 0 0 0 07.5 10 0.01 ug/ml 0 0 0 0 0 0 10 10 0.1 ug/ml 0 0 0 0 0 0 27.5 10 1ug/ml 0 0 0 0 0 0 40 10 10 ug/ml 0 0 0 0 0 0 87 10

Example 15 Antiproliferation Assay Using a BAF3 Cell Line

BaF3 is used to determine if Zcyto20 has anti-proliferative properties.Baby hamster kidney (BHK) cells are stably transfected with anexpression vector containing the CMV promoter plus intron A upstream ofthe Zcyto20cDNA or an unrelated cDNA, called Zα30, using BRLlipofectamine. Stably transfected cells are seeded in a cell factory inserum free media and allowed to grow for three days before conditionedmedia is harvested and concentrated in a 5K filter to 10x. Concentratedconditioned medium samples are stored at 4° C.

The following assay is used to test for anti-proliferation of BaF3. In a96 well plate, eight 1:2 serial dilutions are made of growth media alone(RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mML-glutamine), or murine IL-3 (starting at 50 pg/ml in growth medium)with final volume of 100 μl. Fifty microliters of the following areadded to both growth media alone or mIL-3 diluted lanes: humaninterferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium),human interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growthmedium), murine interferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted ingrowth medium), murine interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/mldiluted in growth medium), Zcyto20(at 2.5×, 0.5×, or 0.1×), and murineZα30 (at 2.5×, 0.5×, or 0.1×).

The BaF3 cell line is washed three times in growth medium, pellets areresuspended in growth medium, cells are counted and diluted in growthmedium to 5,000 cells/50 μl. Fifty microliters of diluted cells are thenadded to each dilution of samples. Assay plates are incubated in a 37°C. incubator for three to four days. Twenty microliters of Alomar blueare then added to each well and the plate are incubated overnight at 37°C. The plates are read on the fluorescent plate reader at excitationwavelength of 544 and emission wavelength 590.

Example 16 Signaling Via Interferon-Response Pathway

Interaction of type 1 interferons with their specific receptor leads toinduction of a number of genes responsible for theirantiviral/antiproliferative activity. These include 2′-5′ oligoadenylatesynthetase (2-5A synthetase), double-stranded RNA dependent Pkr kinase(Pkr), phospholipid scramblase, and intercellular adhesion molecule-1(ICAM-1). Induction of genes with as yet unknown function, such as a 56kDa interferon stimulated gene product (ISG-56k), also occurs. Todetermine if some or all of these genes are induced upon treatment ofcells with zcyto20, human Daudi B lymphoid cells were treated for 72hours with conditioned medium from Sf9 cells infected with baculovirusexpressing zcyto20. Conditioned medium from Sf9 cells infected withwild-type baculovirus was used as a negative control. Followingtreatment cells were collected and lysed for isolation of total RNA. 1microgram of total RNA was converted to cDNA using reverse transcriptaseand used as a template for polymerase chain reaction usingoligonucleotide primers specific for the human interferon-stimulatedgenes described above. Oligonucleotide primers for humanglycerol-3-phosphate dehydrogenase (G3PDH) were used as a non-interferonstimulated gene control. The results show clear induction of ISG-56k,Pkr, 2-5A synthetase and phospholipid scramblase following treatment ofcells with zcyto20. No induction was seen for ICAM-1 or thenon-interferon stimulated gene control, G3PDH.

Example 17 Identification of Zcytor19 as a Receptor for Zcyto21

A: COS Cell Transfection and Secretion Trap

Biotinylated zcyto21 was tested for binding to known or orphan cytokinereceptors. The pZP7 expression vectors containing cDNAs of cytokinereceptors (including human IFNαR1, IFNβR2, IFNαR1, IFNβR2, IL-10R,CRF2-4, ZcytoR7, DIRS1, Zcytor19, and Tissue Factor) were transfectedinto COS cells, and the binding of biotinylated zcyto20 to transfectedCOS cells was carried out using the secretion trap assay describedbelow. Positive binding in this assay showed receptor-ligand pairs.

COS Cell Transfections

The COS cell transfections were performed as follows: COS cells wereplated (1×10⁵ cells/well) on fibronectin coated, 12-well, tissue cultureplates (Becton Dickinson, Bedford, Mass.) and incubated at 37° C.overnight. Cytokine receptor DNA (0.75 μg) was mixed with 50 μL serumfree DMEM media (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mgtransferrin, 2.5 mg insulin, 1 g selenium and 5 mg fetuin in 500 mlDMEM), then mixed with 5 μl Lipofectamine™ (Invitrogen, Carlsbad,Calif.) in 45 μl serum free DMEM media, and incubated at roomtemperature for 30 minutes. An additional 400 μl serum free DMEM mediawas added. The cells were rinsed with serum free DMEM, and 500 μL of theDNA mixture was added. The cells were incubated for 5 hours at 37° C.,at which time an additional 500 μL 20% FBS DMEM media (100 ml FBS, 55 mgsodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) was added and thecells were incubated overnight.

Secretion Trap Assay

The secretion trap was performed as follows: Media was aspirated andcells were rinsed twice with 1% BSA in PBS. Cells were blocked for 1hour with TNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NENRenaissance TSA-Direct Kit, NEN Life Science Products, Boston, Mass.) inH₂O. The cells were incubated for 1 hour with 3 μg/ml biotinylatedzcyto21 protein (Example 27) in TNB. Cells were then washed 3 times with1% BSA in PBS and were incubated for another hour with 1:300 dilutedStreptavidin-HRP (NEN kit) in TNB. Again cells were washed 3 times with1% BSA in PBS, and then fixed for 15 minutes with 1.8% Formaldehyde inPBS. Cells were then washed 3 times with TNT (0.1 M Tris-HCL, 0.15MNaCl, and 0.05% Tween-20 in H₂O).

Positive binding was detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (NEN kit), incubated for 4.5 minutes, and washedwith TNT. Cells were preserved with Vectashield Mounting Media (VectorLabs Burlingame, Calif.) diluted 1:5 in TNT. Cells were visualized usinga FITC filter on fluorescent microscope.

Positive binding was detected on cells transfected with human zcytor19cDNA and incubated with biotinylated zcyto21. None of the othertransfected receptors bound zcyto21, and zcytor19 did not bind a controlbiotinylated protein. These data indicate that zcytor19 is a receptorfor zcyto21.

Further experiments have shown positive binding between both human andmouse Zcytor19 with biotinylated zcyto21. Positive binding was alsodetected on cells transfected with human zcytor19 cDNA and incubatedwith biotinylated zcyto20, and zcyto24.

Example 18 Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to determine thefunctional interaction of zcyto20, zcyto21, zcyto22, zcyto24, andzcyto25 with zcytor19. Human embryonal kidney (HEK) cells aretransfected with a reporter plasmid containing an interferon-stimulatedresponse element (ISRE) driving transcription of a luciferase reportergene in the presence or absence of pZP7 expression vectors containingcDNAs for class II cytokine receptors (including human DIRS1, IFNαR1,IFNαR2 and Zcytor19 (SEQ ID NOS:23 and 26)). Luciferase activityfollowing stimulation of transfected cells with class II ligands(including zcyto20 (SEQ ID NO:1), zcyto21 (SEQ ID NO:4), zcyto22 (SEQ IDNO:6), zcyto10, huIL10 and huIFNa-2a) reflects the interaction of theligand with transfected and native cytokine receptors on the cellsurface. The results and methods are described below.

Cell Transfections

293 HEK cells were transfected as follows: 700,000 293 cells/well (6well plates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and1 microgram pIRES2-EGFP DNA (Clontech,) were added to 9 microlitersFugene 6 reagent (Roche Biochemicals) in a total of 100 microlitersDMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNAwas not included. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with dilutions (in DMEM+0.5% FBS) of the following class IIligands; zcyto20, zcyto21, zcyto22, zcyto10, huIL10 and huIFNa-2a.Following a 4-hour incubation at 37° C., the cells were lysed, and therelative light units (RLU) were measured on a luminometer after additionof a luciferase substrate. The results obtained are shown as the foldinduction of the RLU of the experimental samples over the medium alonecontrol (RLU of experimental samples/RLU of medium alone=foldinduction). Table 14 shows that zcyto20, zcyto21 and zcyto22 induce ISREsignaling in 293 cells transfected with ISRE-luciferase giving a 15 to17-fold induction in luciferase activity over medium alone. The additionof zcytor19 DNA to the transfection mix results in a 6 to 8-fold furtherinduction in ISRE signaling by zcyto20, zcyto21 and zcyto22 giving a 104to 125-fold total induction. None of the other transfected class IIcytokine receptor DNAs resulted in increased ISRE signaling. Theseresults indicate that zcyto20, zcyto21 and zcyto22 functionally interactwith the zcytor19 cytokine receptor. Table 13 also shows that huIFNa-2acan induce ISRE signaling in ISRE-luciferase transfected 293 cellsgiving a 205-fold induction of luciferase activity compared to mediumalone. However, the addition of zcytor19 DNA to the transfection leadsto an 11-fold reduction in ISRE-signaling (compared to ISRE-luciferaseDNA alone), suggesting that zcytor19 over-expression negatively effectsinterferon signaling, in contrast to the positive effects of zcytor19over-expression on zcyto20, zcyto21 and zcyto22 signaling.

TABLE 13 Interferon Stimulated Response Element (ISRE) Signaling ofTransfected 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Ligand ISRE-Luc. ISRE-Luc./Zcytor19 Zcyto20 (125 ng/ml) 15125 Zcyto21 (125 ng/ml) 17 108 Zcyto22 (125 ng/ml) 17 104 HuIFNa-2a (100ng/ml) 205 18 Zcyto10 (125 ng/ml) 1.3 1 HuIL10 (100 ng/ml) 1 0.5

Example 19 Identification of IL10Rb (CRF2-4) as a Receptor Subunit forZcytor19

A: IL10Rb Neutralizing Antibody Inhibits ISRE Signaling:

A signal transduction reporter assay was used to determine thefunctional interaction of zcyto20, zcyto21, and zcyto22 with zcytor19and IL10Rb (CRF2-4). Human embryonal kidney (HEK) cells or humanembryonal kidney (HEK) cells stably overexpressing human zcytoR19 weretransfected with a reporter plasmid containing an interferon-stimulatedresponse element (ISRE) driving transcription of a luciferase reporter.Luciferase activity following stimulation of transfected cells withclass II ligands (including zcyto20, zcyto21, zcyto22 and huIFNa-2a) inthe presence or absence of a neutralizing antibody to IL10Rb (CRF2-4)reflects the interaction of the ligand with cytokine receptors on thecell surface. The results and methods are described below.

Cell Transfections:

To produce 293 HEK cells stably overexpressing human zcytoR19, 293 cellswere transfected as follows: 300,000 293 cells/well (6 well plates) wereplated approximately 6 h prior to transfection in 2 milliliters DMEM+10%fetal bovine serum. Per well, 2 micrograms of a pZP7 expression vectorcontaining the cDNA of human zcytoR19 (SEQ ID NO:23) was added to 6microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100microliters DMEM. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Forty-eight hours later the transfected cellswere placed under 2 microgram/milliliter puromicin selection. Puromicinresistant cells were carried as a population of cells.

The 293 HEK cells (wild type or overexpressing human zcytoR19) weretransfected as follows: 700,000 293 cells/well (6 well plates) wereplated approximately 18 h prior to transfection in 2 millilitersDMEM+10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA(Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech) were added to 6microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100microliters DMEM. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays:

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37 degrees in DMEM+0.5% FBS, transfected cellswere pretreated with a neutralizing polyclonal goat antibody to IL10Rb(2.5 micrograms/ml for zcyto21; 8 micrograms/ml for zcyto20 and zcyto22,R&D Systems) or PBS for 1 hour at 37 C. Human embryonal kidney (HEK)cells stably overexpressing human zcytoR19 were also pretreated with anon-neutralizing polyclonal goat antibody to IFNAR1 (8 micrograms/ml,R&D Systems) as an antibody control for experiments involving zcyto20and zcyto22. Pretreated cells were stimulated with dilutions (inDMEM+0.5% FBS) of the following class II ligands; zcyto20, zcyto21, orzcyto22. As a control, huIFNa-2a was run in each experiment. Following a4-hour incubation at 37 degrees, the cells were lysed, and the relativelight units (RLU) were measured on a luminometer after addition of aluciferase substrate. The results obtained are shown as the foldinduction of the RLU of the experimental samples over the medium alonecontrol (RLU of experimental samples/RLU of medium alone=foldinduction).

Tables 14 and 15 show that induction of ISRE signaling by zcyto20 isinhibited by pretreatment of wild type 293 cells or 293 cellsoverexpressing human zcytoR19 with a neutralizing antibody to IL10Rb. Noor little inhibition is seen of huIFNa-2a induction of ISRE signaling.These results indicate that zcyto20 requires interaction with IL10Rb(CRF2-4) for maximal induction of ISRE signaling and that the receptorfor zcyto20 is the heterdimeric combination of zcytoR19 and IL10Rb(CRF2-4).

TABLE 14 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type293 Cells Following Class II Cytokine Stimulation (Fold Induction)Zcyto20 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizingneutralizing (ng/ml) Zcyto20 Antibody HuIFNa-2a Antibody 100 8.4 0.8 152102 10 4 0.9 160 117 1 1 0.9 90 69 0.1 1 1 12 6 0.01 1 0.8 1.2 1 0 1 1 11

TABLE 15 IL10Rb Inhibition of ISRE Signaling of Transfected zcytoR19-overexpressing 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Zcyto20 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentrationneutralizing neutralizing (ng/ml) Zcyto20 Antibody HuIFNa-2a Antibody100 91 60 16 16 10 97 23 14 15 1 68 1.3 8 8.4 0.1 6 1.1 1.5 1.9 0.01 1.11.2 1.2 1.3 0 1 1 1 1

Tables 16 and 17 show that ISRE signaling by zcyto21 is inhibited bypretreatment of wild type 293 cells or 293 cells overexpressing humanzcytoR19 with a neutralizing antibody to IL10Rb. No inhibition is seenof huIFNa-2a induction of ISRE signaling. These results indicate thatzcyto21 requires interaction with IL10Rb (CRF2-4) for maximal inductionof ISRE signaling and that the receptor for zcyto21 is the heterodimericcombination of zcytoR19 and IL10Rb (CRF2-4).

TABLE 16 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type293 Cells Following Class II Cytokine Stimulation (Fold Induction)Zcyto21 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizingneutralizing (ng/ml) Zcyto21 Antibody HuIFNa-2a Antibody 100 4.1 1.8 3130 10 3.2 1.4 32 31 1 1.5 1.3 16.3 15 0.1 1.1 1.3 1.4 2 0.01 1.2 1.3 1.11.2 0.001 1.2 1.3 0.9 2.1 0 1 1 1 1

TABLE 17 IL10Rb Inhibition of ISR) Signaling of Transfected zcytoR19-overexpressing 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Zcyto21 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentrationneutralizing neutralizing (ng/ml) Zcyto21 Antibody HuIFNa-2a Antibody100 45 31 9 7.7 10 48 28 9 8.5 1 35 5.8 4.3 4.3 0.1 3.5 1 1.4 1.3 0.011.5 1.1 0.9 1.2 0.001 1.1 1 1.2 1 0 1 1 1 1

Tables 18 and 19 show that induction of ISRE signaling by zcyto22 isinhibited by pretreatment of wild type 293 cells or 293 cellsoverexpressing human zcytoR19 with a neutralizing antibody to IL10Rb. Noor little inhibition is seen of huIFNa-2a induction of ISRE signaling.These results indicate that zcyto22 requires interaction with IL10Rb(CRF2-4) for maximal induction of ISRE signaling and that the receptorfor zcyto22 is the heterodimeric combination of zcytoR19 and IL10Rb(CRF2-4).

TABLE 18 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type293 Cells Following Class II Cytokine Stimulation (Fold Induction)Zcyto22 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizingneutralizing (ng/ml) Zcyto22 Antibody HuIFNa-2a Antibody 100 11 1.2 152102 10 8 1 160 117 1 1.8 0.8 90 69 0.1 1.2 0.8 12 6 0.01 0.9 0.9 1.2 1 01 1 1 1

TABLE 19 IL10Rb Inhibition of ISRE Signaling of Transfected zcytoR19-overexpressing 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Zcyto22 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentrationneutralizing neutralizing (ng/ml) Zcyto22 Antibody HuIFNa-2a Antibody100 82 76 16 16 10 97 39 14 15 1 69 2.3 8 8.4 0.1 8.4 1.1 1.5 1.9 0.01 11.3 1.2 1.3 0 1 1 1 1

B: A: Anti-IL10Rb Antibody Blocks Antiviral Activity

An antiviral assay was performed to determine the ability of anti-IL10Rbantibody to block the antiviral activity of zcyto20. The assay wascarried out using 293 HEK cells (wild type or overexpressing humanzcytoR19). On the first day, antibodies (anti-human IL10R beta,anti-human Leptin receptor, R&D Systems) were diluted into cell media at5 micrograms/ml and then plated with 50,000 cells per well into a96-well plate. Following a one-hour incubation at 37° C., zcyto20-CEE(from example 3) (200 ng/ml for wild-type 293 cells, 0.5 ng/ml for 293cells overexpressing human zcytoR19) or human interferon-a-2a (1 ng/mlfor wild-type 293 cells, 100 ng/ml for 293 cells overexpressing humanzcytoR19) were added to the wells and incubated overnight at 37° C. Thenext day, the medium was removed and replaced with medium containingencephalomyocarditis virus (EMCV) at a multiplicity of infection of 0.1.The cells were then incubated at 37° C. overnight. Subsequently, 25 uLof 5 mg/ml Methylthiazoletetrazolium (MTT)(Sigma) were added to eachwell, incubated 2 hours at 37 degrees, and wells were then extractedwith 100 uL extraction buffer (12.5% SDS, 45% DMF). Following overnightincubation at 37° C., the optical density at 570 nM was measured on aSpectromax plate reader (Molecular Devices, CA). Decreased opticaldensity (570 nm) indicates decreased cell survival (loss of antiviralactivity). The optical densities (570 nm) for the different experimentalconditions are shown in Table 20 below. The results indicate thatblocking human IL10 receptor beta specifically neutralizes the antiviralactivity of zcyto20 without effecting interferon-a-2a activity. Thisindicates that human IL10 receptor beta is part of the receptor complex(including human zcytoR19) involved in zcyto20 antiviral activity.

TABLE 20 Optical Density (570 nm) of ECMV-Infected Cytokine-TreatedCells HuzcytoR19- HuzcytoR19- Wild-type Wild-type overexpressingoverexpressing 293 293 293 293 Cells: Anti- Cells: Anti- Cells: Anti-Cells: Anti- Cytokine IL10Rb LeptinR IL10Rb LeptinR Zcyto20- .94 .88 .95.24 CEE HuIFNa- .58 .4  .18 .05 2a

C: Zcyto20 Zcyto21 and Zcyto22 Signaling is Enhanced by Coexpression ofZcytoR19 and IL10Rb:

A signal transduction reporter assay was used to determine thefunctional interaction of zcyto20, zcyto21 and zcyto22 with zcytor19 andIL 10Rb (CRF2-4). Hamster kidney (BHK) cells were transfected with areporter plasmid containing an interferon-stimulated response element(ISRE) driving transcription of a luciferase reporter gene in thepresence or absence of pZP7 expression vectors containing cDNAs forclass II cytokine receptors Zcytor19 and IL10Rb (CRF2-4). Luciferaseactivity following stimulation of transfected cells with class IIligands (including zcyto20, zcyto21 and zcyto22) reflects theinteraction of the ligand with transfected and native cytokine receptorson the cell surface. The results and methods are described below.

Cell Transfections

BHK-570 cells were transfected as follows: 200,000 BHK cells/well (6well plates) were plated approximately 5 h prior to transfection in 2milliliters DMEM+5% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and1 microgram pIRES2-EGFP DNA (Clontech) were added to 9 microlitersFugene 6 reagent (Roche Biochemicals) in a total of 100 microlitersDMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNAwas not included. This transfection mix was added 30 minutes later tothe pre-plated BHK cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with dilutions (in DMEM+0.5% FBS) of zcyto20, zcyto21,zcyto22, zcyto24, and zcyto25 ligands. Following a 4-hour incubation at37 degrees, the cells were lysed, and the relative light units (RLU)were measured on a luminometer after addition of a luciferase substrate.The results obtained are shown as the fold induction of the RLU of theexperimental samples over the medium alone control (RLU of experimentalsamples/RLU of medium alone=fold induction). Table 21 shows thatzcyto20, zcyto21 and zcyto22 induce ISRE signaling in BHK cellstransfected with ISRE-luciferase and zcytoR19 in a dose-dependentmanner. The addition of IL10Rb (CRF2-4) DNA to the transfection mixresults in a half-maximal induction of signaling at a 10-100 fold lowercytokine dose. No response was seen with ISRE transfection alone. Theseresults show that the ability of zcyto20, zcyto21 and zcyto22 to signalthrough the interferon stimulated response element is enhanced bycoexpression of zcytoR19 and IL10Rb (CRF2-4) indicating that thereceptor for zcyto20, zcyto21 and zcyto22 is the heterodimericcombination of zcytoR19 and IL10Rb (CRF2-4).

TABLE 21 Interferon Stimulated Response Element (ISRE) Signaling ofTransfected BHK Cells Following Class II Cytokine Stimulation (FoldInduction) zcyto20/ zcyto21/ zcyto22/ cells cells cells zcyto20/transfected zcyto21/ transfected zcyto22/ transfected cells with cellswith cells with Class II transfected zcytoR19 transfected zcytoR19transfected zcytoR19 ligand with and with and with and ConcentrationzcytoR19 IL10Rb zcytoR19 IL10Rb zcytoR19 IL10Rb (ng/ml) alone (CRF2-4)alone (CRF2-4) alone (CRF2-4) 1000 2.25 2.1 3.3 2.2 1.8 2.2 100 2.2 2.62.6 2.5 2 2.2 10 2.1 2.4 2.4 2.6 1.9 2.7 1 1.3 2.5 2 2.5 1.5 2.7 0.11.25 2.1 1.4 2.2 1.1 2.4 0.01 1.2 1.6 1.4 1.6 1.2 1.7 0.001 1.4 1.5 1.31.3 1.2 1.3 0 1 1 1 1 1 1

Example 20 Construction of Mammalian Expression Vectors that ExpressZcytor19 Soluble Receptors: Zcytor19CEE Zcytor19CFLG zcytor19CHIS andZcytor19-Fc4

An expression vector is prepared for the expression of the soluble,extracellular domain of the zcytor19 polypeptide, pC4zcytor19CEE,wherein the construct is designed to express a zcytor19 polypeptidecomprised of the predicted initiating methionine and truncated adjacentto the predicted transmembrane domain, and with a C-terminal Glu-Glu tag(SEQ ID NO: 16).

A zcytor19 DNA fragment comprising the zcytor19 extracellular orcytokine binding domain of zcytor19 described herein, is created usingPCR, and purified using standard methods. The excised DNA is subclonedinto a plasmid expression vector that has a signal peptide, e.g., thenative zcytor19 signal peptide, and attaches a Glu-Glu tag (SEQ IDNO:16) to the C-terminus of the zcytor19 polypeptide-encodingpolynucleotide sequence. Such a mammalian expression vector contains anexpression cassette having a mammalian promoter, multiple restrictionsites for insertion of coding sequences, a stop codon and a mammalianterminator. The plasmid can also have an E. coli origin of replication,a mammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene and the SV40 terminator.

Restriction digested zcytor19 insert and previously digested vector areligated using standard molecular biological techniques, andelectroporated into competent cells such as DH10B competent cells (GIBCOBRL, Gaithersburg, Md.) according to manufacturer's direction and platedonto LB plates containing 50 mg/ml ampicillin, and incubated overnight.Colonies are screened by restriction analysis of DNA prepared fromindividual colonies. The insert sequence of positive clones is verifiedby sequence analysis. A large scale plasmid preparation is done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process is used to prepare the zcytor19 soluble receptors witha C-terminal his tag, composed of 6 His residues in a row; and aC-terminal FLAG® tag (SEQ ID NO:42), zcytor19CFLAG. To construct theseconstructs, the aforementioned vector has either the C-HIS or the FLAG®tag in place of the glu-glu tag (SEQ ID NO: 16).

An expression vector, zcytor19/Fc4/pzmp20, was prepared to express aC-terminally Fc4 tagged soluble version of zcytor19 (human zcytor19-Fc4)in BHK cells. A fragment of zcytor19 cDNA that includes thepolynucleotide sequence from extracellular domain of the zcytor19receptor was fused in frame to the Fc4 polynucleotide sequence (SEQ IDNO:43) to generate a zcytor19-Fc4 fusion. The pzmp20 vector is amammalian expression vector that contains the Fc4 polynucleotidesequence and a cloning site that allows rapid construction of C-terminalFc4 fusions using standard molecular biology techniques.

A 630 base pair fragment was generated by PCR, containing theextracellular domain of human zcytor19 with BamHI and Bgl2 sites codedon the 5′ and 3′ ends, respectively. This PCR fragment was generatedusing primers ZC37967 (SEQ ID NO:44) and ZC37972 (SEQ ID NO:45) byamplification from human brain cDNA library. The PCR reaction conditionswere as follows: 30 cycles of 94° C. for 20 seconds, and 68° C. for 2minutes; 1 cycle at 68° C. for 4 minutes; followed by a 10° C. soak. Thefragment was digested with BamHI and Bgl2 restriction endonucleases andsubsequently purified by 1% gel electrophoresis and band purificationusing QiaQuick gel extraction kit (Qiagen). The resulting purified DNAwas ligated for 5 hours at room temperature into a pzmp20 vectorpreviously digested with Bgl2 containing Fc4 3′ of the Bgl2 sites.

One μl of the ligation mix was electroporated in 37 μl DH10Belectrocompetent E. coli (Gibco) according to the manufacturer'sdirections. The transformed cells were diluted in 400 μl of LB media andplated onto LB plates containing 100 μg/ml ampicillin. Clones wereanalyzed by restriction digests and positive clones were sent for DNAsequencing to confirm the sequence of the fusion construct.

Example 21 Mammalian Expression Human Zcytor19 Soluble Receptor:Zcytor19/Fc4

BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75 tissue cultureflasks and allowed to grow to approximately 50 to 70% confluence at 37°C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL,Gaithersburg, Md.), 5% fetal bovine serum, 1 mM L-glutamine (JRHBiosciences, Lenea, Kans.), 1 mM sodium pyruvate (Gibco BRL)). The cellswere then transfected with the plasmid zcytor19/Fc4/pzmp20 (Example 4B)using Lipofectamine™ (Gibco BRL), in serum free (SF) media formulation(DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1%L-glutamine and 1% sodium pyruvate). Ten μg of the plasmid DNAzcytor19/Fc4/pzmp20 (Example 4B) was diluted into a 15 ml tube to atotal final volume of 500 μl with SF media. 50 μl of Lipofectamine wasmixed with 450 μl of SF medium. The Lipofectamine mix was added to theDNA mix and allowed to incubate approximately 30 minutes at roomtemperature. Four ml of SF media was added to the DNA:Lipofectaminemixture. The cells were rinsed once with 5 ml of SF media, aspirated,and the DNA:Lipofectamine mixture was added. The cells were incubated at37° C. for five hours, and then 5 ml of DMEM/10% FBS media was added.The flask was incubated at 37° C. overnight after which time the cellswere split into the selection media (DMEMJFBS media from above with theaddition of 1 μM methotrexate (Sigma Chemical Co., St. Louis, Mo.) in150 mm plates at 1:2, 1:10, and 1:50. Approximately 10 dayspost-transfection, one 150 mm plate of 1 μM methotrexate resistantcolonies was trypsinized, the cells were pooled, and one-half of thecells were replated in 10 μM methotrexate; to further amplify expressionof the zcytor19/Fc4 protein. A conditioned-media sample from this poolof amplified cells was tested for expression levels using SDS-PAGE andWestern analysis.

Single clones expressing the soluble receptors can also isolated,screened and grown up in cell culture media, and purified using standardtechniques. Moreover, CHO cells are also suitable cells for suchpurposes.

Example 22 Assessing Zcytor19 Receptor Heterodimerization Using ORIGENAssay

Soluble zcytor19 receptor is biotinylated by reaction with a five-foldmolar excess of sulfo-NHS-LC-Biotin (Pierce, Inc., Rockford, Ill.)according to the manufacturer's protocol. Soluble zcytor19 receptor andanother soluble receptor subunit, for example, soluble class II cytokinereceptors, for example, CRF2-4 (SEQ ID NO:40), are labeled with a fivefold molar excess of Ru—BPY—NHS (Igen, Inc., Gaithersburg, Md.)according to manufacturer's protocol. The biotinylated andRu—BPY—NHS-labeled forms of the soluble zcytor19 receptor can berespectively designated Bio-zcytor19 receptor and Ru-zcytor19; thebiotinylated and Ru—BPY—NHS-labeled forms of the other soluble receptorsubunit can be similarly designated. Assays are carried out usingconditioned media or using purified ligands.

For initial soluble receptor binding characterization, the cytokinesmentioned above, or conditioned medium, are tested to determine whetherthey can mediate homodimerization of zcytor19 receptor and if they canmediate the heterodimerization of zcytor19 receptor with the solublereceptor subunits described above. To do this, 50 μl of conditionedmedia or TBS-B containing purified cytokine, is combined with 50 μl ofTBS-B (20 mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing e.g.,400 ng/ml of Ru-zcytor19 receptor and Bio-zcytor19, or 400 ng/ml ofRu-zcytor19 receptor and e.g., Bio-CRF2-4, or 400 ng/ml of e.g.,Ru—CRF2-4 and Bio-zcytor19. Following incubation for one hour at roomtemperature, 30 μg of streptavidin coated, 2.8 mm magnetic beads (Dynal,Inc., Oslo, Norway) are added and the reaction incubated an additionalhour at room temperature. 200 μl ORIGEN assay buffer (Igen, Inc.,Gaithersburg, Md.) is then added and the extent of receptor associationmeasured using an M8 ORIGEN analyzer (Igen, Inc.).

Example 23 Construct for Generating a Zcytor19 Receptor Heterodimer

A vector expressing a secreted human zcytor19 heterodimer is constructedby fusing the extracellular cytokine-binding domain of zcytor19 to theheavy chain of IgG gamma 1 (IgGγ1), and the extracellular portion of theheteromeric cytokine receptor subunit (E.g., class II cytokinereceptors, for example, CRF2-4) is fused to a human kappa light chain(human k light chain).

a. Construction of IgG Gamma 1 and Human κ Light Chain Fusion Vectors

The heavy chain of IgGγ1 is cloned into the Zem229R mammalian expressionvector (ATCC deposit No. 69447) such that any desired cytokine receptorextracellular domain having a 5′ EcoRI and 3′ NheI site can be cloned inresulting in an N-terminal extracellular domain-C-terminal IgGγ1 fusion.The IgGγ1 fragment used in this construct is made by using PCR toisolate the IgGγ1 sequence from a Clontech human Fetal Liver cDNAlibrary as a template. PCR products are purified using methods describedherein and digested with MluI and EcoRI (Boerhinger-Mannheim), ethanolprecipitated and ligated with oligos that comprise an MluI/EcoRI linker,into Zem229R previously digested with and EcoRI using standard molecularbiology techniques disclosed herein.

The human κ light chain is cloned in the Zem228R mammalian expressionvector (ATCC deposit No. 69446) such that any desired cytokine receptorextracellular domain having a 5′ EcoRI site and a 3′ KpnI site can becloned in resulting in a N-terminal cytokine extracellulardomain-C-terminal human κ light chain fusion. As a KpnI site is locatedwithin the human κ light chain sequence, a special primer is designed toclone the 3′ end of the desired extracellular domain of a cytokinereceptor into this KpnI site: The primer is designed so that theresulting PCR product contains the desired cytokine receptorextracellular domain with a segment of the human κ light chain up to theKpnI site. This primer preferably comprises a portion of at least 10nucleotides of the 3′ end of the desired cytokine receptor extracellulardomain fused in frame 5′ to the human kappa light chain. The human κlight chain fragment used in this construct is made by using PCR toisolate the human κ light chain sequence from the same Clontech humanFetal Liver cDNA library used above. PCR products are purified usingmethods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with theMluI/EcoRI linker described above, into Zem228R previously digested withand EcoRI using standard molecular biology techniques disclosed herein.

b. Insertion of Zcytor19 Receptor or Heterodimeric Subunit ExtracellularDomains into Fusion Vector Constructs

Using the construction vectors above, a construct having zcytor19 fusedto IgGγ1 is made. This construction is done by PCRing the extracellulardomain or cytokine-binding domain of zcytor19 receptor described hereinfrom a prostate cDNA library (Clontech) or activated lymphocyte cDNAlibrary using standard methods, and oligos that provide EcoRI and NheIrestriction sites. The resulting PCR product is digested with EcoRI andNheI, gel purified, as described herein, and ligated into a previouslyEcoRI and NheI digested and band-purified Zem229R/IgGγ1 described above.The resulting vector is sequenced to confirm that the zcytor19/IgG gamma1 fusion (zcytor19/Ch1 IgG) is correct.

A separate construct having a heterodimeric cytokine receptor subunit,i.e., CRF2-4, extracellular domain fused to κ light is also constructedas above. The cytokine receptor/human κ light chain construction isperformed as above by PCRing from, e.g., a lymphocyte cDNA library(Clontech) using standard methods, and oligos that provide EcoRI andKpnI restriction sites. The resulting PCR product is digested with EcoRIand KpnI and then this product is ligated into a previously EcoRI andKpnI digested and band-purified Zem228R/human κ light chain vectordescribed above. The resulting vector is sequenced to confirm that thecytokine receptor subunit/human κ light chain fusion is correct.

c. Co-Expression of the Zcytor19 and Heterodimeric Cytokine ReceptorSubunit Extracellular Domain

Approximately 15 μg of each of vectors above, are co-transfected intomammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells are selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants is selected again in 10 μm of MTX and 0.5 mg/ml G418 for10 days.

The resulting pool of doubly selected cells is used to generate protein.Three Factories (Nunc, Denmark) of this pool are used to generate 10 Lof serum free conditioned medium. This conditioned media is passed overa 1 ml protein-A column and eluted in about 10, 750 microliterfractions. The fractions having the highest protein concentration arepooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzedmaterial is submitted for amino acid analysis (AAA) using routinemethods.

d. Reconstitution of Zcytor19 Receptor In Vitro

To identify components involved in the zcytor19-signaling complex,receptor reconstitution studies are performed as follows. For example,BHK 570 cells (ATCC No. CRL-10314) transfected, using standard methodsdescribed herein, with a luciferase reporter mammalian expression vectorplasmid serve as a bioassay cell line to measure signal transductionresponse from a transfected zcytor19 receptor complex to the luciferasereporter in the presence of zcytor19 Ligand. BHK cells would be used inthe event that BHK cells do not endogenously express the zcytor19receptor. Other cell lines can be used. An exemplary luciferase reportermammalian expression vector is the KZ134 plasmid which is constructedwith complementary oligonucleotides that contain STAT transcriptionfactor binding elements from 4 genes. A modified c-fos Sis inducibleelement (m67SIE, or hSIE) (Sadowski, H. et al., Science 261:1739-1744,1993), the p21 SIE1 from the p21 WAFI gene (Chin, Y. et al., Science272:719-722, 1996), the mammary gland response element of the β-caseingene (Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991), anda STAT inducible element of the Fcg R1 gene, (Seidel, H. et al., Proc.Natl. Acad. Sci. 92:3041-3045, 1995). These oligonucleotides containAsp718-XhoI compatible ends and are ligated, using standard methods,into a recipient firefly luciferase reporter vector with a c-Fospromoter (Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998)digested with the same enzymes and containing a neomycin selectablemarker. The KZ134 plasmid is used to stably transfect BHK, or BaF3cells, using standard transfection and selection methods, to make aBHK/KZ134 or BaF3/KZ134 cell line respectively.

The bioassay cell line is transfected with zcytor19 receptor alone, orco-transfected with zcytor19 receptor along with one of a variety ofother known receptor subunits. Receptor complexes include but are notlimited to zcytor19 receptor only, various combinations of zcytor19receptor with class II cytokine receptors, for example,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511) receptors. Each independent receptor complex cell line isthen assayed in the presence of cytokine-conditioned media or purifiedcytokines and luciferase activity measured using routine methods. Theuntransfected bioassay cell line serves as a control for the backgroundluciferase activity, and is thus used as a baseline to compare signalingby the various receptor complex combinations. The conditioned medium orcytokine that binds the zcytor19 receptor in the presence of the correctreceptor complex, is expected to give a luciferase readout ofapproximately 5 fold over background or greater.

As an alternative, a similar assay can be performed wherein the aBaf3/zcytor19 cell line isco-transfected as described herein andproliferation is measured, using a known assay such as a standard AlamarBlue proliferation assay.

Example 24 Binding of Ligands to Soluble Receptors

The binding of the ligands (zcyto20, zcyto21, zcyto22, zcyto24, andzcyto25) to soluble receptors can be assayed using an iodo-bead labelingmethod. For example, ¹²⁵I labeled zcyto21-CEE is labeled (1.2×10⁷CPM/ml; 1.5 ng/ul; and 8.6×10⁶ CPM/ug).

Fifty nanograms of the ¹²⁵I labeled zcyto21-cEE (See Example 3) (399,600CPM) is combined with 1000 ng of cold zcytor19/Fc4 homodimer receptor,1000 ng cold zcytor19/CRF2-4 heterodimer receptor, or 1000 ng of acontrol Class II cytokine receptor/Fc4 receptor as a control with about10,000 ng of cold zcyto21 as a competitor. Samples are incubated for 2hours at 4° C., after which 30 ul protein-G (Zymed San Francisco,Calif.) is added to each sample. Samples are incubated for 1 hour at 4°C., and washed 3 times with PBS. Radioactivity of the washed protein-Gis measured in a gamma counter (Packard Instruments, Downers Grove,Ill.).

Example 25 Flow Cytometry Staining of Human Monocytes with Zcyto20 andZcyto21-Biotin

Peripheral blood leukocytes (PBLs) were isolated by Ficoll Hypaque(Amersham, Sweden) separation from heparinized human blood. The PBLswere cultured at 37° C. in standard media at a density of 1×10^(e)6cells per milliliter in 6-well tissue culture plates. Followingovernight incubation, the PBLs were harvested and stained withbiotinylated zcyto20-cee and zcyto21-cee (See Example 18) at aconcentration of 10 ug/ml. Staining was detected withPhycoerythrin-labeled streptavidin (Pharmingen, Calif., USA) that wasprepared at a dilution of 1:1000. Following staining the PBLs were fixedin 2% Paraformaldehyde, and read on a Facscaliber (Becton Dickinson, SanDiego, Calif.). The data was analyzed using Cellquest software (BectonDickinson). Results indicate that both biotinylated zcyto20-cee andzcyto21-cee stain cells in the myeloid gate of peripheral bloodleukocytes. Cells in the lymphoid gate do not bind zcyto20-cee andzcyto21-cee.

Example 26 Zcyto21-CEE Effects on Expression of Activation Markers onPBLs

Peripheral blood leukocytes (PBLs) were isolated by Ficoll Hypaqueseparation from heparinized human blood. The PBLs were then stimulatedwith purified protein or media controls from the following: 1)zcyto21-CEE (2 ug/ml); 2) zcyto21-cee (1 ug/ml); 3) Media alone; 4)A141F negative control protein (2 ug/ml); or 5) IFN-alpha-A (1 ng/ml)(PBL Biomedical N.J., USA.). The stimulated PBLs were incubated at acell density of 1×10^(e)6 cells per milliliter in at 37° C. with 5% CO2.Cultures were harvested at 24 and 48 hours and stained for activationmarkers.

The PBLs were washed with PBS and then blocked with normal mouse IgG inFacs buffer (HBSS+2% normal goat serum, 2% BSA, 0.2% NaN3), followed bystaining with antibodies for the following markers: CD19, CD14, CD3,HLA-DR, CD54, HLA-ABC (Pharmingen, Calif., USA and Immunotech, France).Cells were washed and then fixed in 2% Paraformaldehyde before beinganalyzed on a Facscaliber (Becton Dickinson, Calif., USA). The resultingdata were analyzed using Cellquest software (Becton Dickinson, Calif.,USA).

Results indicated an increase in surface CD54 (ICAM) expression onmonocytes at 24 and 48 hours with zcyto21-cee stimulation, compared tothe media alone control. Stimulation with zcyto21-cee also resulted inan increase in Major histocompatibility complex I expression on B-cellsat 24 hours and an increase in MHCI on both B-cells and Monocytes at 48hours.

Example 27 Biotinylation of Ligands

Zcyto21CEE were biotinylated by following a modified protocol by PierceChemical Company for sulfo-NHS-LC-Biotin. Two hundred and fiftymicrograms of zcyto21CEE (in 0.5 ml PBS) was added to 8.4 ul ofS—NHS-biotin stock (84 ug), and incubated at room temperature for 2hours with rocking. Following the incubation step, 20 ul 2M Tris HCl(pH8) was added, and the mixture was incubated for 20 minutes at roomtemperature with rocking. The biotinylated ligand mixtures were thenstored at 4° C. Zcyto20CEE, zcyto22CEE and zcyto24CEE were prepared inessentially the same manner.

Example 28 Expression of Zcytor19 by Northern Analysis

Northern blots were probed to determine the tissue distribution ofzcytor19. A human zcytor19 cDNA fragment was obtained using PCR withgene specific primers, 5′ ZC40285 as shown in SEQ ID NO: 21; and 3′ ZC40286, as shown in SEQ ID NO: 22. The template was cloned human zcytor19cDNA. (SEQ ID NO: 23) The PCR fragment was gel purified, and ˜25 ng waslabeled with P³²α-dCTP using the Prime-It® RmT random prime labeling kit(Stratagene, LaJolla, Calif.).

The following Northern blots (Clontech, Palo Alto, Calif.) were probedfor mRNA expression of zcytor19: (1) a human cancer cell line blot C,which contains RNA samples from each of the following cancer cell lines:promyelocytic leukemia HL-60, HELA S3, chronic myelogenous leukemiak-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma RAJI,colorectal adenocarcinoma SW480, lung carcinoma A549, and melanomaG-361; (2) a human MTN H blot, which contains mRNA from the followingtissues: heart, whole brain, placenta, lung, liver, skeletal muscle,kidney, and pancreas; (3) a human MTN H3 which contains mRNA from thefollowing tissues: stomach, thyroid, spinal cord, lymph node, trachea,adrenal gland, and bone marrow; and (4) a human MTN H4, which containsmRNA from the following tissues: spleen, thymus, prostate testis,uterus, small intestine, colon, and peripheral blood leukocytes.Hybridizations were all performed in ULTRAhyb™ UltrasensitiveHybridization Buffer (Ambion, Austin, Tex.) according the manufacturer'srecommendations, which the exception that an additional 0.2 mg/ml salmonsperm DNA was added to the hybridization and prehybridization buffers tolower non-specific hybridization. Following hybridization, non-specificradioactive signal was removed by treating the blots with 0.1×SSC/0.5%SDS at 50° C. The blots were exposed using BioMax MR Film andintensifying screens (Eastman Kodak, Rochester, N.Y.), per themanufacturer's recommendations for 3 days.

Expression of a ˜4.5 kb transcript was in greatest in heart, skeletalmuscle, pancreas and prostate tissue, in addition to in the Burkitt'slymphoma (RAJI) cell line. Lower levels were seen in multiple othertissues. In addition, there was an ˜2 kb transcript which was generallyless abundant than the larger transcript, but also present in many ofthe tissues and cell lines. Testis tissue, in addition to having the 2and 4.5 kb transcripts, may also have ˜4 kb and 1.4 kb transcripts.Adrenal gland demonstrated equal levels of expression of the 4.5 kb and2 kb transcripts.

Example 29 Expression of Zcytor19 by In Situ Analysis

Specific human tissues were isolated and screened for zcytor19expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included normal andcarcinoma colon, cervical carcinoma, endometrial carcinoma, normal andcarcinoma ovary, normal and neoplasmic skin, fetal liver, lung, heartand MFH (muscle sarcoma). The tissues were fixed in 10% bufferedformalin and blocked in paraffin using standard techniques. Tissues weresectioned at 4 to 8 microns. Tissues were prepared using a standardprotocol. Briefly, tissue sections were deparaffinized with Histo-Clear®(National Diagnostics, Atlanta, Ga.) and then dehydrated with ethanol.Next they were digested with Proteinase K (50 μg/ml) (BoehringerDiagnostics, Indianapolis, Ind.) at 37° C. for 2 to 7 minutes. This stepwas followed by acetylation and re-hydration of the tissues.

One in situ probe was designed against the human zcytor19 (variant x1)sequence (INC7128744, as shown in SEQ ID NO: 25), containing the 3′UTRof zcytor19 using standard methods. T7 RNA polymerase was used togenerate an antisense probe. The probe was labeled using an In Vitrotranscription System (Riboprobe® in vitro Transcription System, Promega,Madison, Wis.) as per manufacturer's instruction, except that the probesdigoxigenin was used instead of radiolabeled rCTP and that the water wasadjusted to accommodate the reduced volume of the rNTP's. In situhybridization was performed with a digoxigenin-labeled zcytor19 probe(above). The probe was added to the slides at a concentration of 1 to 5pmol/ml for 12 to 16 hours at 60° C. Slides were subsequently washed in2×SSC and 0.1×SSC at 55° C. The signals were amplified using TSA™(Tyramide Signal Amplification; PerkinElmer Life Sciences Inc., Boston,Mass.) and visualized with VECTOR Red substrate kit (VectorLaboratories, Burlingame, Calif.) as per manufacturer's instructions.The slides were then counter-stained with hematoxylin.

Signals were observed in several tissues tested: In colon carcinomatissues, weak signal was observed in carcinoma cells and a few immuneinfiltrations. However, there was no positive signal observed in thenormal colon and intestine, including cells in lamina propria,epithelium, immune nodules and peripheral ganglia nerve cells. Incervical carcinoma tissues, there is weak signal in carcinoma cells andsome cells in the immune nodules. In endometrial carcinoma tissues, weaksignals present in the carcinoma cells. In normal uterus tissues, nopositive signal was observed. In ovarian carcinoma samples, somecarcinoma cells are weakly positive. In normal ovary samples, someendothelium of capillaries and epithelium of large follicles may beweakly positive. In the skin carcinoma sample, the cancerous granularepithelium is strongly positive, while no positive signal is observed inthe normal skin. In fetal liver, signal is observed in a mixedpopulation of mononuclear cells in sinusoid spaces. In lung, zcytor19appears to be positive in type II alveolar epithelium. Occasionallybronchial epithelium may also be weakly positive. Macrophage-likemononuclear cells in the interstitial tissue are also positive. Inheart, myocytes are negative while some circulating mononuclear cellsare positive for zcytor19. In one of the samples, endothelium of thevessels may be weakly positive. Other tissues tested including a MFH(muscle sarcoma) sample and a Kaposi's sarcoma skin sample. There is noconclusive positive signal in these tissues.

Example 30 Human Zcytor19 Expression Based on RT-PCR Analysis ofStimulated Versus Non-Stimulated Cells

Gene expression of zcytor19 was examined using RT-PCR analysis of thefollowing cell types: Hela, 293, Daudi, CD14+, U937, and HL-60.

First-strand cDNA synthesis from total RNA was carried out using acommercially available first-strand synthesis system for RT-PCR(Invitrogen life technologies, Carlsbad, Calif.). The subsequent PCRreactions were set up using zcytor19×1 (SEQ ID NO:23) and zcytor19×2(SEQ ID NO:28) specific oligo primers ZC40288 (SEQ ID NO:58) and ZC40291(SEQ ID NO:59) which yield a 806 bp and 892 bp product, respectively,Qiagen HotStarTaq DNA Polymerase and Buffer, (Qiagen, Inc., Valencia,Calif.), GeneAmp dNTPs (Applied Biosystems, Foster City, Calif.),RediLoad™ dye (Research Genetics, Inc., Huntville, Ala.) and 2 μlfirst-strand cDNA (10% of the first-strand reaction) from the respectivecell types. The PCR cycler conditions were as follows: an initial 1cycle 15 minute denaturation at 95° C., 35 cycles of a 45 seconddenaturation at 94° C., 1 minute annealing at 63° C. and 1 minute and 15second extension at 72° C., followed by a final 1 cycle extension of 7minutes at 72° C. The reactions were separated by electrophoresis on a2% agarose gel (EM Science, Gibbstown, N.J.) and visualized by stainingwith ethidium bromide.

Bands of the correct size were seen in Hela±IFN-beta (only the 892 bpband), 293+Parental Adv, Daudi±IFN-beta, Daudi±IFN-alpha, CD14+activated, HL-60 activated. No band was observed in CD14+ resting, U937resting and activated, and HL-60 resting. These results show inductionof zcytoR19 expression upon activation or differentiation of monocytesor monocyte cell lines.

Example 31 Stimulation of an NFKB Reporter in RAW Cells

The ability of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 to signalthrough the NF kappa beta signal transduction pathway was tested using amouse monocyte/macrophage reporter cell line. This cell line wasgenerated by transducing RAW264.7 cells with the KZ170 retroviralreporter containing NF kappa beta response elements drivingtranscription of a luciferase reporter gene.

Initial reporter assays testing zcyto20, zcyto21, zcyto22, zcyto24 andzcyto25 activity were done using the 293 transient transfectedconditioned media described for use in the antiviral assays.RAW264.7/KZ170 cells were harvested and plated at a density of 50,000cells per well in 96-well plates. The cells were incubated overnight at37° C. in RPMI+10% FBS. On the following day, media was removed from theadherent cells and undiluted zcyto20-25 conditioned media or dilutionsof zcyto20-25 conditioned media (diluted into RPMI+0.1% BSA) were addedto the cells. Following a 5-hour incubation at 37° C. the cells werelysed, and read on a luminometer, after addition of a luciferasesubstrate. The results were analyzed by comparing relative light units(RLU) of zcyto20-25 conditioned media to relative light units ofnon-transfected cell conditioned media. Undiluted zcyto20-25 conditionedmedia induced luciferase expression 4-9 fold higher than undilutednon-transfected cell conditioned media. These results indicate thatzcyto20-25 are able to signal via the NF kappa beta signaling pathway ina mouse monocyte/macrophage cell line.

As a follow up, conditioned media from Sf9 cells infected withbaculovirus expressing zcyto20, zcyto21 or zcyto22 were used in thereporter assays. Wild type baculovirus was used as a negative control.The production of the baculovirus constructs and conditioned media wasdescribed above.

The results of the RAW264.7 NFkb-reporter assay using thebaculovirus-derived conditioned media were similar to that using the 293transient transfected conditioned media. Baculovirus-derived conditionedmedia containing zcyto20-22 induced luciferase expression in a dosedependent manner, while the corresponding control conditioned medium didnot.

Example 32 In Vivo Results

The toxicity and biological activity of zcyto24 was compared to anotherClass II cytokine, and a parental adenovirus vector, as well as innon-injected mice. Four groups of 8 C57B16 mice (female, 9 weeks of age)were injected as follows:

Group 1: Injected with Adzcyto24 at 1×10¹¹ particles per mouse;

Group 2: Injected with a Class II cytokine (Adzcyto) at 1×10¹¹particles;

Group 3: Parental adenovirus vector (Adzpar) at 1×10¹¹ particles; and

Group 4: Untreated

Temperature transponders were placed on the mice on Day −1, and viruswas injected Day 0. Cage-wise food intake and body weight were monitoredevery 5 Days, and blood was sampled on Day 10 (0.25 ml max) for assaysincluding CBC and Abbot blood analyzer. All mice were sacrificed on Day20.

Adzcyto24 (Group 1) treated mice were bled at Day 10 and the sera weretested for the presence of zcyto24 bioactivity. A viral assaydemonstrated significant antiviral activity at a maximal dilution of1:500 for each and every mouse in the Adzcyto24 group. This correspondsto approximately 160 ng/ml of purified zcyto24CEE. An antiviral assay todetect mouse interferons detected no activity in the Adzcyto24 group. Abioassay using a reporter with an ISRE also detected significant zcyto24activity in the sera of the Adzcyto24 injected mice but not the othergroups.

Temperature probes revealed that the mean body temperature for Group 2decreased by more than 5 degrees C. by Day 10 while the mean temp forthe Adzcyto24 (Group 1) group had decreased by more than 2 degrees C.Control groups displayed less than a 1 degree change in temperaturethroughout the experiment.

The Adzcyto24 (Group 1) mice displayed a slight increase in weightduring the 20 Day experiment, equivalent to the control groups (Groups 3and 4). Group 2 mice lost weight: The mean weight for Group 2 decreasedby ˜8% by Day 10.

Abbot blood analyzer analysis of blood obtained on Day 10 and Day 20revealed significant changes in the leukocyte counts in Groups 1 and 2.FIG. 1 shows a pronounced increase in monocyte counts in Adzcyto24treated mice relative to the other groups. Monocyte counts are 2.77times higher in Adzcyto24 injected mice versus parental vector (Adzpar)injected mice. By Day 20 monocyte counts decreased somewhat, but werestill significantly elevated relative to the parental vector injectedmice. FIG. 2 shows that injection with adenovirus encoding a Class IIcytokine (Adzcyto) but not with an adenovirus encoding zcyto24 leads toincreased neutrophil counts on Day 10.

To verify the identity of the cell types detected by the Abbot bloodanalyzer flow cytometry was performed with lineage-specific MAbs onblood from Day 20 of the experiment. FIG. 3 shows the % of CD11bpositive cells, i.e. monocytes in the peripheral blood of each mouse.Plotting the mean values for each group, FIG. 4 reveals a significantincrease in the percent of monocytes in the group injected withadenovirus infected with Zycyto24 versus the group infected withparental vector (p=0.05) This correlates with the changes previouslyobserved with the Abbot blood analyzer.

Analysis of the same blood samples with a MAb specific for granulocytes(GR-1) revealed no significant increase in the percentage ofgranulocytes (i.e. primarily neutrophils) for the Adzcyto24 injectedmice. The PBL were also stained with MAbs specific to B cells (B220) andno significant differences were observed in the Adzcyto24 injected micerelative to the other groups.

Notably the chills and weight loss associated with Adzcyto injectionwere not evident with Adzcyto24. The apparent elevation in monocytesdetected by the Abbot blood analyzer at Days 10 and 20 were confirmed byflow cytometry of Day 20 blood with lineage-specific MAbs. A distinctincrease in the percentage of monocytes in the PBL of mice injected withAdzcyto24 appears to be a unique activity either directly or indirectlymediated by zcyto24 in the context of an adenoviral infection. Zcyto24expressed at significant levels in the Adzcyto24 injected mice is likelypromoting an antiviral response by either recruiting and mobilizingmonocytes from peripheral tissues or by stimulating production ofmonocytes from bone marrow or liver derived progenitor cells. Thisevidence suggests that monocytes/macrophages are activated by zcyto24and zcyto21.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A fusion protein comprising a polypeptide having at least 95%sequence identity with amino acid residues 22-205 of SEQ ID NO:2 and anaffinity tag, wherein the polypeptide binds a receptor as shown in SEQID NOs:24, 27 or 29 as a monomeric or homodimeric receptor or a receptoras shown in SEQ ID NOs:24, 27 or 29 in combination with SEQ ID NO:41 asa heterodimeric receptor.
 2. The fusion protein of claim 1 wherein thepolypeptide comprises amino acid residues 22-205 of SEQ ID NO:2.
 3. Thefusion protein of claim 1 wherein the polypeptide comprises amino acidresidues 1-205 of SEQ ID NO:2.
 4. The fusion protein of claim 1 whereinthe affinity tag is protein A, glutathione S transferase, Glu-Glu tag,or a FLAG peptide. 5-8. (canceled)
 9. The fusion protein of claim 1wherein the polypeptide has at least 96% sequence identity with aminoacid residues 22-205 of SEQ ID NO:2.
 10. The fusion protein of claim 1wherein the polypeptide has at least 97% sequence identity with aminoacid residues 22-205 of SEQ ID NO:2.
 11. The fusion protein of claim 1wherein the polypeptide has at least 98% sequence identity with aminoacid residues 22-205 of SEQ ID NO:2.
 12. The fusion protein of claim 1wherein the polypeptide has at least 99% sequence identity with aminoacid residues 22-205 of SEQ ID NO:2.
 13. A fusion protein comprising apolypeptide comprising amino acid residues 22-205 of SEQ ID NO:7 and anaffinity tag, wherein the polypeptide binds a receptor as shown in SEQID NOs:24, 27 or 29 as a monomeric or homodimeric receptor or a receptoras shown in SEQ ID NOs:24, 27 or 29 in combination with SEQ ID NO:41 asa heterodimeric receptor.
 14. The fusion protein of claim 13 wherein thepolypeptide comprises amino acid residues 1-205 of SEQ ID NO:7.
 15. Thefusion protein of claim 13 wherein the affinity tag is protein A,glutathione S transferase, Glu-Glu tag, or a FLAG peptide.